Methods providing a therapeutic macromolecule and synthetic nanocarriers comprising immunosuppressant locally and concomitantly to reduce both type 1 and type iv hypersensitivity

ABSTRACT

Disclosed are methods and related compositions for concomitantly, locally administering immunosuppressants and doses of therapeutic macromolecules for reducing Type I and Type IV hypersensitivity.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/269,054, filed May 2, 2014, which claims the benefit under 35 U.S.C.§ 119 of U.S. provisional applications 61/819,517, filed May 3, 2013;61/881,851, filed Sep. 24, 2013; 61/881,913, filed Sep. 24, 2013;61/881,921, filed Sep. 24, 2013; 61/907,177, filed Nov. 21, 2013;61/948,313, filed Mar. 5, 2014; and 61/948,384, filed Mar. 5, 2014, theentire contents of each of which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to methods of administering immunosuppressantsand therapeutic doses of therapeutic macromolecules, and relatedcompositions. The methods have been found to be useful in reducing TypeI and Type IV hypersensitivity as a result of therapeutic macromoleculeadministration. The compositions and methods provided can be used togenerate a tolerogenic immune response in a subject at risk of a localinflammatory reaction to a therapeutic dose of a therapeuticmacromolecule.

BACKGROUND OF THE INVENTION

Therapeutic treatments, such as protein or enzyme replacement therapies,often result in undesired immune responses to the particulartherapeutic, such as local inflammation. Such undesired immune responsesmay be reduced through the use of immunosuppressant drugs. Conventionalimmunosuppressant drugs, however, are broad-acting. Additionally, inorder to maintain immunosuppression, immunosuppressant drug therapy isgenerally a life-long proposition. Unfortunately, the use ofbroad-acting immunosuppressants are associated with a risk of severeside effects, such as tumors, infections, nephrotoxicity and metabolicdisorders. Accordingly, new antigen-specific tolerogenic therapies wouldbe beneficial.

SUMMARY OF THE INVENTION

In one aspect, a method comprising providing a therapeutic dose of atherapeutic macromolecule, wherein the therapeutic macromolecule is notattached to synthetic nanocarriers; providing a composition comprisingsynthetic nanocarriers that, in some embodiments are attached toimmunosuppressants; and locally administering the composition and thetherapeutic dose of the therapeutic macromolecule to a subjectconcomitantly, wherein the subject is at risk of a local inflammatoryreaction due to the administration of the therapeutic dose of thetherapeutic macromolecule, and wherein the local concomitantadministration of the composition and the therapeutic dose of thetherapeutic macromolecule reduces both Type 1 hypersensitivity and TypeIV hypersensitivity in the subject is provided.

In one embodiment of any one of the methods provided herein, the subjectis a naïve subject.

In another embodiment of any one of the methods provided herein, thecomposition and the therapeutic dose of the therapeutic macromoleculeare administered to the same location. In another embodiment of any oneof the methods provided herein, the composition and the therapeutic doseof the therapeutic macromolecule are administered to differentlocations.

In another embodiment of any one of the methods provided herein, theconcomitant local administration is according to a protocol that hasbeen demonstrated to result in a reduction of both Type 1hypersensitivity and Type IV hypersensitivity with the composition andtherapeutic dose of the therapeutic macromolecule, as compared to localadministration of the therapeutic dose of the therapeutic macromoleculein the absence of concomitant local administration of the composition.

In another embodiment of any one of the methods provided herein, themethod further comprises determining the protocol.

In another embodiment of any one of the methods provided herein, themethod further comprises assessing a local inflammatory response in thesubject prior to and/or after the administration.

In another embodiment of any one of the methods provided herein, themethod further comprises assessing Type 1 hypersensitivity and Type IVhypersensitivity in the subject prior to and/or after theadministration.

In another embodiment of any one of the methods provided herein, theadministering is by intradermal, intramuscular or subcutaneousadministration.

In another embodiment of any one of the methods provided herein, themethod further comprises recording a reduction or prevention of a localinflammatory response. In another embodiment of any one of the methodsprovided herein, the method further comprises recording a reduction inboth Type 1 hypersensitivity and Type IV hypersensitivity.

In another embodiment of any one of the methods provided herein, theimmunosuppressant comprises a statin, an mTOR inhibitor, a TGF-βsignaling agent, a corticosteroid, an inhibitor of mitochondrialfunction, a P38 inhibitor, an NF-κB inhibitor, an adenosine receptoragonist, a prostaglandin E2 agonist, a phosphodiesterase 4 inhibitor, anHDAC inhibitor or a proteasome inhibitor. In another embodiment of anyone of the methods provided herein, the mTOR inhibitor is rapamycin.

In another embodiment of any one of the methods provided herein, thetherapeutic macromolecule is a therapeutic protein or a therapeuticpolynucleotide. In another embodiment of any one of the methods providedherein, the therapeutic protein is for protein replacement of proteinsupplementation therapy. In another embodiment of any one of the methodsprovided herein, the therapeutic protein comprises a/an infusible orinjectable therapeutic protein, enzyme, enzyme cofactor, hormone, bloodor blood coagulation factor, cytokine, interferon, growth factor,monoclonal antibody, polyclonal antibody, or protein associated withPompe's disease. In another embodiment of any one of the methodsprovided herein, the infusible or injectable thereapeutic proteincomprises Tocilizumab, alpha-1 antitrypsin, Hematide, albinterferonalfa-2b, Thucin, tesamorelin, ocrelizumab, belimumab, pegloticase,taliglucerase alfa, agalsidase alfa, or velaglucerase alfa. In anotherembodiment of any one of the methods provided herein, the enzymecomprises an ocidforeductase, transferase, hydrolase, lysase, isomeraseor ligase. In another embodiment of any one of the methods providedherein, the enzyme comprises an enzyme for enzyme replacement therapyfor a lysosomal storage disorder. In another embodiment of any one ofthe methods provided herein, the enzyme for replacement therapy for alysosomal storage disorder comprises imiglucerase, a-galactosidase A(a-gal A), agalsidase beta, acid α-glucosidase (GAA), alglucosidasealfa, LUMIZYME, MYOZYME, arylsulfatase B, laronidase, ALDURAZYME,idursulfase, ELAPRASE, arylsulfatase B, pegloticase, pegsiticase orNAGLAZYME. In another embodiment of any one of the methods providedherein, the cytokine comprises a lymphokine, interleukin, chemokine,type 1 cytokine or a type 2 cytokine. In another embodiment of any oneof the methods provided herein, the blood or blood coagulation factorcomprises Factor I, Factor II, tissue factor, Factor V, Factor VII,Factor VIII, Factor IX, Factor X, Factor Xa, Factor XII, Factor XIII,von Willebrand factor, prekallikrein, high-molecular weight kininogen,fibronectin, antithrombin III, heparin cofactor II, protein C, proteinS, protein Z, protein Z-related protease inhibitor (ZPI), plasminogen,alpha 2-antiplasmin, tissue plasminogen activator (tPA), urokinase,plasminogen activator inhibitor-1 (PAI1), plasminogen activatorinhibitor-2 (PAI2), cancer procoagulant or epoetin alfa.

In another embodiment of any one of the methods provided herein, a loadof immunosuppressant attached to the synthetic nanocarriers, on averageacross the synthetic nanocarriers, is between 0.1% and 50%. In anotherembodiment of any one of the methods provided herein, the load isbetween 0.1% and 20%.

In another embodiment of any one of the methods provided herein, thesynthetic nanocarriers comprise lipid nanoparticles, polymericnanoparticles, metallic nanoparticles, surfactant-based emulsions,dendrimers, buckyballs, nanowires, virus-like particles or peptide orprotein particles. In another embodiment of any one of the methodsprovided herein, the synthetic nanocarriers comprise lipidnanoparticles. In another embodiment of any one of the methods providedherein, the synthetic nanocarriers comprise liposomes. In anotherembodiment of any one of the methods provided herein, the syntheticnanocarriers comprise metallic nanoparticles. In another embodiment ofany one of the methods provided herein, the metallic nanoparticlescomprise gold nanoparticles. In another embodiment of any one of themethods provided herein, the synthetic nanocarriers comprise polymericnanoparticles. In another embodiment of any one of the methods providedherein, the polymeric nanoparticles comprise polymer that is anon-methoxy-terminated, pluronic polymer. In another embodiment of anyone of the methods provided herein, the polymeric nanoparticles comprisea polyester, polyester attached to a polyether, polyamino acid,polycarbonate, polyacetal, polyketal, polysaccharide, polyethyloxazolineor polyethyleneimine. In another embodiment of any one of the methodsprovided herein, the polyester comprises a poly(lactic acid),poly(glycolic acid), poly(lactic-co-glycolic acid) or polycaprolactone.In another embodiment of any one of the methods provided herein, thepolymeric nanoparticles comprise a polyester and a polyester attached toa polyether. In another embodiment of any one of the methods providedherein, the polyether comprises polyethylene glycol or polypropyleneglycol.

In another embodiment of any one of the methods provided herein, themean of a particle size distribution obtained using dynamic lightscattering of the synthetic nanocarriers is a diameter greater than 100nm. In another embodiment of any one of the methods provided herein, thediameter is greater than 150 nm. In another embodiment of any one of themethods provided herein, the diameter is greater than 200 nm. In anotherembodiment of any one of the methods provided herein, the diameter isgreater than 250 nm. In another embodiment of any one of the methodsprovided herein, the diameter is greater than 300 nm.

In another embodiment of any one of the methods provided herein, anaspect ratio of the synthetic nanocarriers is greater than 1:1, 1:1.2,1:1.5, 1:2, 1:3, 1:5, 1:7 or 1:10.

In another aspect, a method of manufacturing any one of the compositionsor kits provided herein is provided. In one embodiment, the method ofmanufacturing comprises producing a dose or dosage form of a therapeuticmacromolecule and producing a dose or dosage form of animmunosuppressant. In one embodiment of any one of the methods ofmanufacturing provided, the step of producing a dose or dosage form ofan immunosuppressant comprises attaching the immunosuppressant tosynthetic nanocarriers. In another embodiment of any one of the methodsof manufacturing provided, the method further comprises combining thedose or dosage form of the immunosuppressant and dose or dosage form ofthe therapeutic macromolecule in a kit.

In another aspect, a use of any of the compositions or kits providedherein for the manufacture of a medicament for reducing both Type 1hypersensitivity and Type IV hypersensitivity, in a subject is provided.In one embodiment of any one of the uses provided herein, theimmunosuppressant is attached to synthetic nanocarriers.

In another aspect, any one of the compositions or kits provided hereinmay be for use in any one of the methods provided herein. In oneembodiment, the medicament comprises a dose or dosage form of aimmunosuppressant and a dose or dosage form of a therapeuticmacromolecule. In another embodiment of any one of the compositions orkits provided herein, the immunosuppressant is attached to syntheticnanocarriers.

In another aspect, a method of manufacturing a medicament intended forreducing both Type 1 hypersensitivity and Type IV hypersensitivity, isprovided. In one embodiment, the medicament comprises a dose or dosageform of a immunosuppressant and a dose or dosage form of a therapeuticmacromolecule. In another embodiment of any one of the methods ofmanufacturing provided herein, the immunosuppressant is attached tosynthetic nanocarriers.

BRIEF DESCRIPTION OF FIGURES

FIGS. 1 and 2 show the reduction in IgG titers and pad swelling as aresult of inventive treatments.

FIG. 3 shows a decrease in foot pad swelling (left panel) and decreasein anti-KLH IgG antibodies (right panel) in animals that receivednanocarriers attached to immunosuppressants concomitantly with KLH,indicating the immunosuppressant compositions are able to reducehypersensitivity reactions to a macromolecule.

FIG. 4 shows the titer of anti-OVA IgG antibodies were reduced inanimals that received nanocarriers attached to immunosuppressantsconcomitantly with OVA, indicating the immunosuppressant compositionsare able to reduce hypersensitivity reaction specific to an administeredmacromolecule.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularlyexemplified materials or process parameters as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments of the inventiononly, and is not intended to be limiting of the use of alternativeterminology to describe the present invention.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entiretyfor all purposes.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “a polymer”includes a mixture of two or more such molecules or a mixture ofdiffering molecular weights of a single polymer species, reference to “asynthetic nanocarrier” includes a mixture of two or more such syntheticnanocarriers or a plurality of such synthetic nanocarriers, reference to“a RNA molecule” includes a mixture of two or more such RNA molecules ora plurality of such RNA molecules, reference to “an immunosuppressant”includes a mixture of two or more such materials or a plurality of suchimmunosuppressant molecules, and the like.

As used herein, the term “comprise” or variations thereof such as“comprises” or “comprising” are to be read to indicate the inclusion ofany recited integer (e.g. a feature, element, characteristic, property,method/process step or limitation) or group of integers (e.g. features,element, characteristics, properties, method/process steps orlimitations) but not the exclusion of any other integer or group ofintegers. Thus, as used herein, the term “comprising” is inclusive anddoes not exclude additional, unrecited integers or method/process steps.

In embodiments of any one of the compositions and methods providedherein, “comprising” may be replaced with “consisting essentially of” or“consisting of”. The phrase “consisting essentially of” is used hereinto require the specified integer(s) or steps as well as those which donot materially affect the character or function of the claimedinvention. As used herein, the term “consisting” is used to indicate thepresence of the recited integer (e.g. a feature, element,characteristic, property, method/process step or limitation) or group ofintegers (e.g. features, element, characteristics, properties,method/process steps or limitations) alone.

A. Introduction

The compositions and methods provided herein have been found,surprisingly, to reduce both Type I and Type IV hypersensitivity.Specifically, it has been found that delivering immunosuppressants,preferably attached to synthetic nanocarriers, by local, concomitantadministration with therapeutic doses of therapeutic macromoleculereduces both types of hypersensitivity. Accordingly, the methods andcompositions provided herein are useful in subjects at risk of a localinflammatory response that otherwise would result or be expected toresult when a therapeutic macromolecule is locally administered withoutthe concomitant local administration of the immunosuppressant.Interestingly, the beneficial effects are more pronounced whenadministering higher doses of therapeutic macromolecules than at lowerdoses. The local, concomitant delivery of immunosuppressants withtherapeutic macromolecules, is therefore provided. The methods andcompositions provided herein can be used to reduce Type I and Type IVhypersensitivity and can be used for subjects that are in need oftherapeutic macromolecule therapy, such as by injection for localadministration.

The inventors have unexpectedly and surprisingly discovered that theproblems and limitations noted above can be overcome by practicing theinvention disclosed herein. The present invention is illustrated in theExamples below.

The invention will now be described in more detail below.

B. Definitions

“Administering” or “administration” or “administer” means providing amaterial to a subject in a manner that is pharmacologically useful. Theterm is intended to include “causing to be administered” in someembodiments. “Causing to be administered” means causing, urging,encouraging, aiding, inducing or directing, directly or indirectly, athird another party to administer the material.

“Amount effective” in the context of a composition or dosage form foradministration to a subject refers to an amount of the composition ordosage form that produces one or more desired immune responses in thesubject, for example, the generation of a tolerogenic immune response(e.g., a reduction in or prevention of a local inflammatory response toa therapeutic macromolecule). Therefore, in some embodiments, an amounteffective is the amount of a composition provided herein that producesone or more of these desired immune responses. The amount effective canbe for in vitro or in vivo purposes. For in vivo purposes, the amountcan be one that a clinician would believe may have a clinical benefitfor a subject in need of reducing or preventing a local inflammatoryresponse in a subject as a result of local administration of atherapeutic macromolecule. Preferably, the amount effective is one thatreduces Type I and Type IV hypersensitivity.

Amounts effective can involve reducing the level of an undesired immuneresponse (e.g., a local inflammatory response), although in someembodiments, it involves preventing an undesired immune responsealtogether. Amounts effective can also involve delaying the occurrenceof an undesired immune response. An amount that is effective can also bean amount of a composition provided herein that produces a desiredtherapeutic endpoint or a desired therapeutic result. Amounts effective,preferably, result in a tolerogenic immune response in a subject to anantigen. The achievement of any of the foregoing can be monitored byroutine methods.

In some embodiments of any one of the compositions and methods provided,the amount effective is one in which the desired immune responsepersists in the subject for at least 1 week, at least 2 weeks or atleast 1 month. In other embodiments of any of the compositions andmethods provided, the amount effective is one which produces ameasurable desired immune response, for example, a measurable decreasein an immune response (e.g., to a specific antigen), for at least 1week, at least 2 weeks or at least 1 month.

Amounts effective will depend, of course, on the particular subjectbeing treated; the severity of a condition, disease or disorder; theindividual patient parameters including age, physical condition, sizeand weight; the duration of the treatment; the nature of concurrenttherapy (if any); the specific route of administration and like factorswithin the knowledge and expertise of the health practitioner. Thesefactors are well known to those of ordinary skill in the art and can beaddressed with no more than routine experimentation. It is generallypreferred that a maximum dose be used, that is, the highest safe doseaccording to sound medical judgment. It will be understood by those ofordinary skill in the art, however, that a patient may insist upon alower dose or tolerable dose for medical reasons, psychological reasonsor for virtually any other reason.

In general, doses of the immunosuppressants and/or therapeuticmacromolecules in the compositions of the invention refer to the amountof the immunosuppressants and/or therapeutic macromolecules.Alternatively, the dose can be administered based on the number ofsynthetic nanocarriers that provide the desired amount ofimmunosuppressants and/or antigens.

“Antigen-specific” refers to any immune response that results from thepresence of the antigen, or portion thereof, or that generates moleculesthat specifically recognize or bind the antigen. For example, where theimmune response is antigen-specific antibody production, antibodies areproduced that specifically bind the antigen. In some embodiments, whenthe antigen comprises the therapeutic macromolecule, antigen-specificmay mean therapeutic macromolecule-specific. In embodiments, such aresponse counteracts the therapeutic effects of the therapeuticmacromolecule.

“Assessing an immune response” refers to any measurement ordetermination of the level, presence or absence, reduction, increase in,etc. of an immune response in vitro or in vivo. Such measurements ordeterminations may be performed on one or more samples obtained from asubject. Such assessing can be performed with any of the methodsprovided herein or otherwise known in the art. The assessing may beassessing the reduction, prevention, presence or absence of a localinflammatory response to a therapeutic macromolecule. The assessing maybe assessing the reduction of Type I and Type IV hypersensitivity.

“Attach” or “Attached” or “Couple” or “Coupled” (and the like) means tochemically associate one entity (for example a moiety) with another. Insome embodiments, the attaching is covalent, meaning that the attachmentoccurs in the context of the presence of a covalent bond between the twoentities. In non-covalent embodiments, the non-covalent attaching ismediated by non-covalent interactions including but not limited tocharge interactions, affinity interactions, metal coordination, physicaladsorption, host-guest interactions, hydrophobic interactions, TTstacking interactions, hydrogen bonding interactions, van der Waalsinteractions, magnetic interactions, electrostatic interactions,dipole-dipole interactions, and/or combinations thereof. In embodiments,encapsulation is a form of attaching. In embodiments, therapeuticmacromolecules and immunosuppressants are not attached to one another,meaning that the therapeutic macromolecules and immunosuppressants arenot subjected to a process specifically intended to chemically associateone with another. In embodiments, immunosuppressants are not attached tosynthetic nanocarriers, meaning that the immunosuppressants andsynthetic nanocarriers are not subjected to a process specificallyintended to chemically associate one with another.

“Average”, as used herein, refers to the arithmetic mean unlessotherwise noted.

“Combination”, as applied to two or more materials and/or agents (alsoreferred to herein as the components), is intended to define material inwhich the two or more materials/agents are associated. Components may beseparately identified, e.g. first component, second component, thirdcomponent, etc. The terms “combined” and “combining” in this context areto be interpreted accordingly.

The association of the two or more materials/agents in a combination maybe physical or non-physical. Examples of physically associated combinedmaterials/agents include:

-   -   compositions (e.g. unitary formulations) comprising the two or        more materials/agents in admixture (for example within the same        unit dose);    -   compositions comprising material in which the two or more        materials/agents are chemically/physicochemically linked (for        example by crosslinking, molecular agglomeration or binding to a        common vehicle moiety);    -   compositions comprising material in which the two or more        materials/agents are chemically/physicochemically co-packaged        (for example, disposed on or within lipid vesicles, particles        (e.g. micro- or nanoparticles) or emulsion droplets);    -   pharmaceutical kits, pharmaceutical packs or patient packs in        which the two or more materials/agents are co-packaged or        co-presented (e.g. as part of an array of unit doses); Examples        of non-physically associated combined materials/agents include:    -   material (e.g. a non-unitary formulation) comprising at least        one of the two or more materials/agents together with        instructions for the extemporaneous association of the at least        one compound/agent to form a physical association of the two or        more materials/agents;    -   material (e.g. a non-unitary formulation) comprising at least        one of the two or more materials/agents together with        instructions for combination therapy with the two or more        materials/agents;    -   material comprising at least one of the two or more        materials/agents together with instructions for administration        to a patient population in which the other(s) of the two or more        materials/agents have been (or are being) administered;    -   material comprising at least one of the two or more        materials/agents in an amount or in a form which is specifically        adapted for use in combination with the other(s) of the two or        more materials/agents.

As used herein, the term “combination therapy” is intended to definetherapies which comprise the use of a combination of two or morematerials/agents (as defined above). Thus, references to “combinationtherapy”, “combinations” and the use of materials/agents “incombination” in this application may refer to materials/agents that areadministered as part of the same overall treatment regimen. As such, theposology of each of the two or more materials/agents may differ: eachmay be administered at the same time or at different times. It willtherefore be appreciated that the materials/agents of the combinationmay be administered sequentially (e.g. before or after) orsimultaneously, either in the same pharmaceutical formulation (i.e.together), or in different pharmaceutical formulations (i.e.separately). Simultaneously in the same formulation is as a unitaryformulation whereas simultaneously in different pharmaceuticalformulations is non-unitary. The posologies of each of the two or morematerials/agents in a combination therapy may also differ with respectto the route of administration.

“Concomitantly” means administering two or more materials/agents to asubject in a manner that is correlated in time, preferably sufficientlycorrelated in time so as to provide a modulation in a physiologic orimmunologic response, and even more preferably the two or morematerials/agents are administered in combination. In embodiments,concomitant administration may encompass administration of two or morematerials/agents within a specified period of time, preferably within 1month, more preferably within 1 week, still more preferably within 1day, and even more preferably within 1 hour. In embodiments, thematerials/agents may be repeatedly administered concomitantly; that isconcomitant administration on more than one occasion, as may be providedin the Examples.

“Determining” or “determine” means to ascertain a factual relationship.Determining may be accomplished in a number of ways, including but notlimited to performing experiments, or making projections. For instance,a dose of an immunosuppressant or therapeutic macromolecule may bedetermined by starting with a test dose and using known scalingtechniques (such as allometric or isometric scaling) to determine thedose for administration. Such may also be used to determine a protocolas provided herein. In another embodiment, the dose may be determined bytesting various doses in a subject, i.e. through direct experimentationbased on experience and guiding data. In embodiments, “determining” or“determine” comprises “causing to be determined.” “Causing to bedetermined” means causing, urging, encouraging, aiding, inducing ordirecting or acting in coordination with an entity for the entity toascertain a factual relationship; including directly or indirectly, orexpressly or impliedly.

“Dosage form” means a pharmacologically and/or immunologically activematerial in a medium, carrier, vehicle, or device suitable foradministration to a subject. Any one of the compositions or dosesprovided herein may be in a dosage form.

“Dose” refers to a specific quantity of a pharmacologically and/orimmunologically active material for administration to a subject for agiven time.

“Encapsulate” means to enclose at least a portion of a substance withina synthetic nanocarrier. In some embodiments, a substance is enclosedcompletely within a synthetic nanocarrier. In other embodiments, most orall of a substance that is encapsulated is not exposed to the localenvironment external to the synthetic nanocarrier. In other embodiments,no more than 50%, 40%, 30%, 20%, 10% or 5% (weight/weight) is exposed tothe local environment. Encapsulation is distinct from absorption, whichplaces most or all of a substance on a surface of a syntheticnanocarrier, and leaves the substance exposed to the local environmentexternal to the synthetic nanocarrier.

“Generating” means causing an action, such as a physiologic orimmunologic response (e.g., a tolerogenic immune response) to occur,either directly oneself or indirectly.

“Identifying a subject” is any action or set of actions that allows aclinician to recognize a subject as one who may benefit from themethods, compositions or kits provided herein. Preferably, theidentified subject is one who is in need of a reduction in a localinflammatory response as provided herein, such as a subject in need ofthe reduction in both Type I and Type IV hypersensitivity due to localadministration of therapeutic macromolecules. The action or set ofactions may be either directly oneself or indirectly. In one embodimentof any one of the methods provided herein, the method further comprisesidentifying a subject in need of a method, composition or kit asprovided herein.

“Immunosuppressant” means a compound that causes an APC to have animmunosuppressive effect (e.g., tolerogenic effect) or a T cell or a Bcell to be suppressed. An immunosuppressive effect generally refers tothe production or expression of cytokines or other factors by the APCthat reduces, inhibits or prevents an undesired immune response or thatpromotes a desired immune response, such as a regulatory immuneresponse. When the APC acquires an immunosuppressive function (under theimmunosuppressive effect) on immune cells that recognize an antigenpresented by this APC, the immunosuppressive effect is said to bespecific to the presented antigen. Without being bound by any particulartheory, it is thought that the immunosuppressive effect is a result ofthe immunosuppressant being delivered to the APC, preferably in thepresence of an antigen. In one embodiment, the immunosuppressant is onethat causes an APC to promote a regulatory phenotype in one or moreimmune effector cells. For example, the regulatory phenotype may becharacterized by the inhibition of the production, induction,stimulation or recruitment of antigen-specific CD4+ T cells or B cells,the inhibition of the production of antigen-specific antibodies, theproduction, induction, stimulation or recruitment of Treg cells (e.g.,CD4+CD25highFoxP3+Treg cells), etc. This may be the result of theconversion of CD4+ T cells or B cells to a regulatory phenotype. Thismay also be the result of induction of FoxP3 in other immune cells, suchas CD8+ T cells, macrophages and iNKT cells. In one embodiment, theimmunosuppressant is one that affects the response of the APC after itprocesses an antigen. In another embodiment, the immunosuppressant isnot one that interferes with the processing of the antigen. In a furtherembodiment, the immunosuppressant is not an apoptotic-signalingmolecule. In another embodiment, the immunosuppressant is not aphospholipid.

Immunosuppressants include, but are not limited to, statins; mTORinhibitors, such as rapamycin or a rapamycin analog; TGF-β signalingagents; TGF-β receptor agonists; histone deacetylase inhibitors, such asTrichostatin A; corticosteroids; inhibitors of mitochondrial function,such as rotenone; P38 inhibitors; NF-κβ inhibitors, such as 6Bio,Dexamethasone, TCPA-1, IKK VII; adenosine receptor agonists;prostaglandin E2 agonists (PGE2), such as Misoprostol; phosphodiesteraseinhibitors, such as phosphodiesterase 4 inhibitor (PDE4), such asRolipram; proteasome inhibitors; kinase inhibitors; G-protein coupledreceptor agonists; G-protein coupled receptor antagonists;glucocorticoids; retinoids; cytokine inhibitors; cytokine receptorinhibitors; cytokine receptor activators; peroxisomeproliferator-activated receptor antagonists; peroxisomeproliferator-activated receptor agonists; histone deacetylaseinhibitors; calcineurin inhibitors; phosphatase inhibitors; PI3 KBinhibitors, such as TGX-221; autophagy inhibitors, such as3-Methyladenine; aryl hydrocarbon receptor inhibitors; proteasomeinhibitor I (PSI); and oxidized ATPs, such as P2X receptor blockers.Immunosuppressants also include IDO, vitamin D3, cyclosporins, such ascyclosporine A, aryl hydrocarbon receptor inhibitors, resveratrol,azathiopurine (Aza), 6-mercaptopurine (6-MP), 6-thioguanine (6-TG),FK506, sanglifehrin A, salmeterol, mycophenolate mofetil (MMF), aspirinand other COX inhibitors, niflumic acid, estriol, methotrexate, andtriptolide. In embodiments, the immunosuppressant may comprise any ofthe agents provided herein.

The immunosuppressant can be a compound that directly provides theimmunosuppressive effect on APCs or it can be a compound that providesthe immunosuppressive effect indirectly (i.e., after being processed insome way after administration). Immunosuppressants, therefore, includeprodrug forms of any of the compounds provided herein.

In embodiments of any one of the methods, compositions or kits providedherein, the immunosuppressants provided herein are attached to syntheticnanocarriers. In preferable embodiments, the immunosuppressant is anelement that is in addition to the material that makes up the structureof the synthetic nanocarrier. For example, in one embodiment, where thesynthetic nanocarrier is made up of one or more polymers, theimmunosuppressant is a compound that is in addition and attached to theone or more polymers. As another example, in one embodiment, where thesynthetic nanocarrier is made up of one or more lipids, theimmunosuppressant is again in addition and attached to the one or morelipids. In embodiments, such as where the material of the syntheticnanocarrier also results in an immunosuppressive effect, theimmunosuppressant is an element present in addition to the material ofthe synthetic nanocarrier that results in an immunosuppressive effect.

Other exemplary immunosuppressants include, but are not limited, smallmolecule drugs, natural products, antibodies (e.g., antibodies againstCD20, CD3, CD4), biologics-based drugs, carbohydrate-based drugs,nanoparticles, liposomes, RNAi, antisense nucleic acids, aptamers,methotrexate, NSAIDs; fingolimod; natalizumab; alemtuzumab; anti-CD3;tacrolimus (FK506); cytokines and growth factors, such as TGF-β andIL-10; etc. Further immunosuppressants, are known to those of skill inthe art, and the invention is not limited in this respect.

In embodiments of any one of the methods, compositions or kits providedherein, the immunosuppressant is in a form, such as a nanocrystallineform, whereby the form of the immunosuppressant itself is a particle orparticle-like. In embodiments, such forms mimic a virus or other foreignpathogen. Many drugs have been nanonized and appropriate methods forproducing such drug forms would be known to one of ordinary skill in theart. Drug nanocrystals, such as nanocrystalline rapamycin are known tothose of ordinary skill in the art (Katteboinaa, et al. 2009,International Journal of PharmTech Resesarch; Vol. 1, No. 3; pp 682-694.As used herein a “drug nanocrystal” refers to a form of a drug (e.g., animmunosuppressant) that does not include a carrier or matrix material.In some embodiments, drug nanocrystals comprise 90%, 95%, 98%, or 99% ormore drug. Methods for producing drug nanocrystals include, withoutlimitation, milling, high pressure homogenization, precipitation, spraydrying, rapid expansion of supercritical solution (RESS), Nanoedge®technology (Baxter Healthcare), and Nanocrystal Technology™ (ElanCorporation). In some embodiments, a surfactant or a stabilizer may beused for steric or electrostatic stability of the drug nanocrystal. Insome embodiments the nanocrystal or nanocrytalline form of animmunosuppressant may be used to increase the solubility, stability,and/or bioavailability of the immunosuppressant, particularlyimmunosuppressants that are insoluble or labile. In some embodiments,local administration of a therapeutic dose of a therapeuticmacromolecule with an immunosupporessant in nanocrytalline form reduceslocal inflammation to a similar extent as is achieved by localadministration of a therapeutic dose of a therapeutic macromolecule witha composition comprising synthetic nanocarriers that are attached to theimmunosuppressants.

“Local inflammation” or “local inflammatory response” means anyinflammatory reaction or response that occurs at a site as a result ofthe administration of a therapeutic macromolecule to the site. In anembodiment, the local inflammation is an inflammatory reaction orresponse that occurs at an injection site when a therapeuticmacromolecule is administered by injection. Local inflammation can bemonitored or assessed by any of the following exemplary methods withoutlimitation, scoring of inflammatory symptoms such as redness orswelling; scoring of arthritic sympthosms such as mobility, pain orjoint destruction; scoring of anaphylaxis symptoms such as swelling,blood pressure, shortness of breath; detecting and/or quantifying cellinfiltration by histology, immunohistochemistry, flow cytometry;measuring the concentration of a protein or inflammation-associatedcytokines such as TNF, IL-1 by ELISA, assessing the expression of geneor inflammation-associated genes by transcriptional analysis; measuringactivity of an inflammation-associated cytokine, etc.

“Hypersensitivity” refers to an undesired immune response to, forexample, a therapeutic macromolecule. There are five types ofhypersensitivity classified based on characteristics of the response.“Type I hypersensitivity” can be mediated by antigen-specific antibodiesof the isotype IgE and IgG4. “Type IV hypersensitivity” or “delayed-typehypersensitivy” may be primarily mediated by T cells. In someembodiments, the methods and compositions provided herein reduce bothType I hypersensitivity and Type IV hypersensitivity to a therapeuticmacromolecule in a subject.

“Locally administered” refers to administration to a specific siterather than systemic administration. In some embodiments of any one ofthe methods provided, the therapeutic dose of the therapeuticmacromolecule and the immunosuppressants are administered to the samelocal administration location. In some embodiments of any one of themethods provided, the therapeutic dose of the therapeutic macromoleculeand the immunosuppressants are administered to different localadministration locations.

“Load” when attached to a synthetic nanocarrier, is the amount of theimmunosuppressant attached to the synthetic nanocarrier based on thetotal dry recipe weight of materials in an entire synthetic nanocarrier(weight/weight). Generally, such a load is calculated as an averageacross a population of synthetic nanocarriers. In one embodiment, theload of the immunosuppressant on average across a population ofsynthetic nanocarriers is between 0.0001% and 99%. In one embodiment,the load of the immunosuppressant on average across a population ofsynthetic nanocarriers is between 0.1% and 50%. In another embodiment,the load of the immunosuppressant is between 0.01% and 20%. In a furtherembodiment, the load of the immunosuppressant is between 0.1% and 10%.In still a further embodiment, the load of the immunosuppressant isbetween 1% and 10%. In still a further embodiment, the load is between7% and 20%. In yet another embodiment, the load of the immunosuppressantis at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, atleast 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least6%, at least at least 7%, at least 8%, at least 9%, at least 10%, atleast 11%, at least 12%, at least 13%, at least 14%, at least 15%, atleast 16%, at least 17%, at least 18%, at least 19% at least 20%, atleast 25%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% on average across the populationof synthetic nanocarriers. In yet a further embodiment, the load of theimmunosuppressant is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%,0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19% or 20% on average across the population of syntheticnanocarriers. In some embodiments of the above embodiments, the load ofthe immunosuppressant is no more than 25% on average across a populationof synthetic nanocarriers. In embodiments, the load is calculated as maybe described in the Examples or as otherwise known in the art.

In some embodiments, when the form of the immunosuppressant is itself aparticle or particle-like, such as a nanocrystalline immunosuppressant,the load of immunosuppressant is the amount of the immunosuppressant inthe particles or the like (weight/weight). In such embodiments, the loadcan approach 97%, 98%, 99% or more.

“Maximum dimension of a synthetic nanocarrier” means the largestdimension of a nanocarrier measured along any axis of the syntheticnanocarrier. “Minimum dimension of a synthetic nanocarrier” means thesmallest dimension of a synthetic nanocarrier measured along any axis ofthe synthetic nanocarrier. For example, for a spheroidal syntheticnanocarrier, the maximum and minimum dimension of a syntheticnanocarrier would be substantially identical, and would be the size ofits diameter. Similarly, for a cuboidal synthetic nanocarrier, theminimum dimension of a synthetic nanocarrier would be the smallest ofits height, width or length, while the maximum dimension of a syntheticnanocarrier would be the largest of its height, width or length. In anembodiment, a minimum dimension of at least 75%, preferably at least80%, more preferably at least 90%, of the synthetic nanocarriers in asample, based on the total number of synthetic nanocarriers in thesample, is equal to or greater than 100 nm. In an embodiment, a maximumdimension of at least 75%, preferably at least 80%, more preferably atleast 90%, of the synthetic nanocarriers in a sample, based on the totalnumber of synthetic nanocarriers in the sample, is equal to or less than5 μm. Preferably, a minimum dimension of at least 75%, preferably atleast 80%, more preferably at least 90%, of the synthetic nanocarriersin a sample, based on the total number of synthetic nanocarriers in thesample, is greater than 110 nm, more preferably greater than 120 nm,more preferably greater than 130 nm, and more preferably still greaterthan 150 nm. Aspects ratios of the maximum and minimum dimensions ofsynthetic nanocarriers may vary depending on the embodiment. Forinstance, aspect ratios of the maximum to minimum dimensions of thesynthetic nanocarriers may vary from 1:1 to 1,000,000:1, preferably from1:1 to 100,000:1, more preferably from 1:1 to 10,000:1, more preferablyfrom 1:1 to 1000:1, still more preferably from 1:1 to 100:1, and yetmore preferably from 1:1 to 10:1. Preferably, a maximum dimension of atleast 75%, preferably at least 80%, more preferably at least 90%, of thesynthetic nanocarriers in a sample, based on the total number ofsynthetic nanocarriers in the sample is equal to or less than 3 μm, morepreferably equal to or less than 2 μm, more preferably equal to or lessthan 1 μm, more preferably equal to or less than 800 nm, more preferablyequal to or less than 600 nm, and more preferably still equal to or lessthan 500 nm. In preferred embodiments, a minimum dimension of at least75%, preferably at least 80%, more preferably at least 90%, of thesynthetic nanocarriers in a sample, based on the total number ofsynthetic nanocarriers in the sample, is equal to or greater than 100nm, more preferably equal to or greater than 120 nm, more preferablyequal to or greater than 130 nm, more preferably equal to or greaterthan 140 nm, and more preferably still equal to or greater than 150 nm.Measurement of synthetic nanocarrier dimensions (e.g., effectivediameter) may be obtained, in some embodiments, by suspending thesynthetic nanocarriers in a liquid (usually aqueous) media and usingdynamic light scattering (DLS) (e.g. using a Brookhaven ZetaPALSinstrument). For example, a suspension of synthetic nanocarriers can bediluted from an aqueous buffer into purified water to achieve a finalsynthetic nanocarrier suspension concentration of approximately 0.01 to0.1 mg/mL. The diluted suspension may be prepared directly inside, ortransferred to, a suitable cuvette for DLS analysis. The cuvette maythen be placed in the DLS, allowed to equilibrate to the controlledtemperature, and then scanned for sufficient time to acquire a stableand reproducible distribution based on appropriate inputs for viscosityof the medium and refractive indicies of the sample. The effectivediameter, or mean of the distribution, is then reported. Determining theeffective sizes of high aspect ratio, or non-spheroidal, syntheticnanocarriers may require augmentative techniques, such as electronmicroscopy, to obtain more accurate measurements. “Dimension” or “size”or “diameter” of synthetic nanocarriers means the mean of a particlesize distribution, for example, obtained using dynamic light scattering.

“Non-methoxy-terminated polymer” means a polymer that has at least oneterminus that ends with a moiety other than methoxy. In someembodiments, the polymer has at least two termini that ends with amoiety other than methoxy. In other embodiments, the polymer has notermini that ends with methoxy. “Non-methoxy-terminated, pluronicpolymer” means a polymer other than a linear pluronic polymer withmethoxy at both termini. Polymeric nanoparticles as provided herein cancomprise non-methoxy-terminated polymers or non-methoxy-terminated,pluronic polymers.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” means a pharmacologically inactive material used together witha pharmacologically active material to formulate the compositions.Pharmaceutically acceptable excipients comprise a variety of materialsknown in the art, including but not limited to saccharides (such asglucose, lactose, and the like), preservatives such as antimicrobialagents, reconstitution aids, colorants, saline (such as phosphatebuffered saline), and buffers.

“Protocol” means a pattern of administering to a subject and includesany dosing regimen of one or more substances to a subject. Protocols aremade up of elements (or variables); thus a protocol comprises one ormore elements. Such elements of the protocol can comprise dosingamounts, dosing frequency, routes of administration, dosing duration,dosing rates, interval between dosing, combinations of any of theforegoing, and the like. In some embodiments, such a protocol may beused to administer one or more compositions of the invention to one ormore test subjects. Immune responses in these test subjects can then beassessed to determine whether or not the protocol was effective ingenerating a desired or desired level of an immune response ortherapeutic effect. Any therapeutic and/or immunologic effect may beassessed. One or more of the elements of a protocol may have beenpreviously demonstrated in test subjects, such as non-human subjects,and then translated into human protocols. For example, dosing amountsdemonstrated in non-human subjects can be scaled as an element of ahuman protocol using established techniques such as alimetric scaling orother scaling methods. Whether or not a protocol had a desired effectcan be determined using any of the methods provided herein or otherwiseknown in the art. For example, a sample may be obtained from a subjectto which a composition provided herein has been administered accordingto a specific protocol in order to determine whether or not specificimmune cells, cytokines, antibodies, etc. were reduced, generated,activated, etc. In preferable embodiments, the reduction, prevention,presence or absence of local inflammation is determined. In even morepreferable embodiments, the reduction, prevention, presence or absenceof Type I and Type IV hypersensitivity is determined. Useful methods fordetecting the presence and/or number of immune cells include, but arenot limited to, flow cytometric methods (e.g., FACS), ELISpot,proliferation responses, cytokine production, and immunohistochemistrymethods. Antibodies and other binding agents for specific staining ofimmune cell markers, are commercially available. Such kits typicallyinclude staining reagents for antigens that allow for FACS-baseddetection, separation and/or quantitation of a desired cell populationfrom a heterogeneous population of cells. In embodiments, a number ofcompositions as provided herein are administered to another subjectusing one or more or all or substantially all of the elements of whichthe protocol is comprised. In some embodiments, the protocol has beendemonstrated to result in a reduction or prevention of a localinflammatory response with the composition and the therapeutic dose ofthe therapeutic macromolecules when locally and concomitantlyadministered as provided herein.

“Providing” means an action or set of actions that an individualperforms that supply a needed item or set of items or methods forpracticing of the present invention. The action or set of actions may betaken either directly oneself or indirectly.

“Providing a subject” is any action or set of actions that causes aclinician to come in contact with a subject and administer a compositionprovided herein thereto or to perform a method provided hereinthereupon. Preferably, the subject is one who is in need ofantigen-specific tolerance or reduction or prevention of localinflammation to a therapeutic macromolecule. The action or set ofactions may be taken either directly oneself or indirectly. In oneembodiment of any one of the methods provided herein, the method furthercomprises providing a subject.

“Recording” means noting, or causing directly or indirectly activitiesin the expectation that such noting would take place, in any written orelectronic form, that a method or composition provided herein achieved areduction in or prevention local inflammation to a therapeuticmacromolecule. In embodiments, any one of the methods provided hereinincludes a step of recording a reduction in Type I and Type IVhypersensitivity. In some embodiments, the recording occurs when atreatment is administered to a subject according to a method as providedherein or at some point thereafter. “Written form”, as used herein,refers to any recordation on a medium such as paper. “Electronic form”,as used herein, refers to any recordation on electronic media. Any oneof the methods provided herein can further comprise a step of recordinga therapeutic and/or immune response in a subject receiving a treatmentaccording to a method provided herein.

“Subject” means animals, including warm blooded mammals such as humansand primates; avians; domestic household or farm animals such as cats,dogs, sheep, goats, cattle, horses and pigs; laboratory animals such asmice, rats and guinea pigs; fish; reptiles; zoo and wild animals; andthe like. “Naïve subject” refers to a subject that has not yet receiveda composition comprising or a therapeutic macromolecule as describedherein.

“Synthetic nanocarrier(s)” means a discrete object that is not found innature, and that possesses at least one dimension that is less than orequal to 5 microns in size. Albumin nanoparticles are generally includedas synthetic nanocarriers, however in certain embodiments the syntheticnanocarriers do not comprise albumin nanoparticles. In embodiments,synthetic nanocarriers do not comprise chitosan. In other embodiments,synthetic nanocarriers are not lipid-based nanoparticles. In furtherembodiments, synthetic nanocarriers do not comprise a phospholipid.

A synthetic nanocarrier can be, but is not limited to, one or aplurality of lipid-based nanoparticles (also referred to herein as lipidnanoparticles, i.e., nanoparticles where the majority of the materialthat makes up their structure are lipids), polymeric nanoparticles,metallic nanoparticles, surfactant-based emulsions, dendrimers,buckyballs, nanowires, virus-like particles (i.e., particles that areprimarily made up of viral structural proteins but that are notinfectious or have low infectivity), peptide or protein-based particles(also referred to herein as protein particles, i.e., particles where themajority of the material that makes up their structure are peptides orproteins) (such as albumin nanoparticles) and/or nanoparticles that aredeveloped using a combination of nanomaterials such as lipid-polymernanoparticles. Synthetic nanocarriers may be a variety of differentshapes, including but not limited to spheroidal, cuboidal, pyramidal,oblong, cylindrical, toroidal, and the like. Synthetic nanocarriersaccording to the invention comprise one or more surfaces. Exemplarysynthetic nanocarriers that can be adapted for use in the practice ofthe present invention comprise: (1) the biodegradable nanoparticlesdisclosed in U.S. Pat. No. 5,543,158 to Gref et al., (2) the polymericnanoparticles of Published US Patent Application 20060002852 to Saltzmanet al., (3) the lithographically constructed nanoparticles of PublishedUS Patent Application 20090028910 to DeSimone et al., (4) the disclosureof WO 2009/051837 to von Andrian et al., (5) the nanoparticles disclosedin Published US Patent Application 2008/0145441 to Penades et al., (6)the protein nanoparticles disclosed in Published US Patent Application20090226525 to de los Rios et al., (7) the virus-like particlesdisclosed in published US Patent Application 20060222652 to Sebbel etal., (8) the nucleic acid attached virus-like particles disclosed inpublished US Patent Application 20060251677 to Bachmann et al., (9) thevirus-like particles disclosed in WO2010047839A1 or WO2009106999A2, (10)the nanoprecipitated nanoparticles disclosed in P. Paolicelli et al.,“Surface-modified PLGA-based Nanoparticles that can EfficientlyAssociate and Deliver Virus-like Particles” Nanomedicine. 5(6):843-853(2010), (11) apoptotic cells, apoptotic bodies or the synthetic orsemisynthetic mimics disclosed in U.S. Publication 2002/0086049, or (12)those of Look et al., Nanogel-based delivery of mycophenolic acidameliorates systemic lupus erythematosus in mice” J. ClinicalInvestigation 123(4):1741-1749(2013). In embodiments, syntheticnanocarriers may possess an aspect ratio greater than 1:1, 1:1.2, 1:1.5,1:2, 1:3, 1:5, 1:7, or greater than 1:10.

Synthetic nanocarriers according to the invention that have a minimumdimension of equal to or less than about 100 nm, preferably equal to orless than 100 nm, do not comprise a surface with hydroxyl groups thatactivate complement or alternatively comprise a surface that consistsessentially of moieties that are not hydroxyl groups that activatecomplement. In a preferred embodiment, synthetic nanocarriers accordingto the invention that have a minimum dimension of equal to or less thanabout 100 nm, preferably equal to or less than 100 nm, do not comprise asurface that substantially activates complement or alternativelycomprise a surface that consists essentially of moieties that do notsubstantially activate complement. In a more preferred embodiment,synthetic nanocarriers according to the invention that have a minimumdimension of equal to or less than about 100 nm, preferably equal to orless than 100 nm, do not comprise a surface that activates complement oralternatively comprise a surface that consists essentially of moietiesthat do not activate complement. In embodiments, synthetic nanocarriersexclude virus-like particles. In embodiments, synthetic nanocarriers maypossess an aspect ratio greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5,1:7, or greater than 1:10.

A “therapeutic dose” refers to a dose of a therapeutic macromoleculethat achieves a desired pharmacological effect upon administration to asubject. Generally, therapeutic doses can be determined by a clinician.

A “therapeutic macromolecule” refers to any protein, carbohydrate, lipidor nucleic acid that may be administered to a subject and have atherapeutic effect. In some embodiments, administration of thetherapeutic macromolecule to a subject may result in an undesired immuneresponse, including local inflammation when locally administered. Insome embodiments, administration of a therapeutic macromoleculeconcomitantly with an immunosuppressant can enhance the therapeuticeffectiveness of the therapeutic macromolecule, such as by reducingundesired immune responses thereto. In some embodiments, the therapeuticmacromolecule may be a therapeutic polynucleotide or therapeuticprotein.

“Therapeutic polynucleotide” means any polynucleotide orpolynucleotide-based therapy that may be administered to a subject andhave a therapeutic effect. Such therapies include gene silencing.Examples of such therapy are known in the art, and include, but are notlimited to, naked RNA (including messenger RNA, modified messenger RNA,and forms of RNAi). Examples of other therapeutic polynucleotides areprovided elsewhere herein. Therapeutic polynucleotides may be producedin, on or by cells and also may be obtained using cell free or fullysynthetic from in vitro methods. Subjects, therefore, include anysubject that is in need of treatment with any of the foregoing. Suchsubject include those that will receive any of the foregoing.

A “therapeutic protein” refers to any protein or protein-based therapythat may be administered to a subject and have a therapeutic effect.Such therapies include protein replacement and protein supplementationtherapies. Such therapies also include the administration of exogenousor foreign proteins, antibody therapies, and cell or cell-basedtherapies. Therapeutic proteins comprise, but are not limited to,enzymes, enzyme cofactors, hormones, blood clotting factors, cytokines,growth factors, monoclonal antibodies, antibody-drug conjugates, andpolyclonal antibodies. Examples of other therapeutic proteins areprovided elsewhere herein. Therapeutic proteins may be produced in, onor by cells and may be obtained from such cells or administered in theform of such cells. In embodiments, the therapeutic protein is producedin, on or by mammalian cells, insect cells, yeast cells, bacteria cells,plant cells, transgenic animal cells, transgenic plant cells, etc. Thetherapeutic protein may be recombinantly produced in such cells. Thetherapeutic protein may be produced in, on or by a virally transformedcell. Subjects, therefore, include any subject that is in need oftreatment with any of the foregoing. Such subject include those thatwill receive any of the foregoing.

“Undesired immune response” refers to any undesired immune response thatresults from exposure to an antigen, promotes or exacerbates a disease,disorder or condition provided herein (or a symptom thereof), or issymptomatic of a disease, disorder or condition provided herein. Suchimmune responses generally have a negative impact on a subject's healthor is symptomatic of a negative impact on a subject's health. Undesiredimmune responses include a local inflammatory response. In someembodiments, the undesired immune response includes Type I and Type IVhypersensitivity.

C. Compositions

Provided herein are compositions comprising immunosuppressants andtherapeutic doses of therapeutic macromolecules, and related methods andkits. Such compositions, kits, and methods are useful for reducing thegeneration of undesired immune responses and promoting the generation oftolerogenic immune responses that are specific to therapeuticmacromolecules. The compositions can be locally and concomitantlyadministered to subjects in which a local inflammatory response occursor is expected to occur. Such subjects include those that are in need oftreatment with a therapeutic macromolecule.

A wide variety of synthetic nanocarriers can be used according to theinvention. In some embodiments, synthetic nanocarriers are spheres orspheroids. In some embodiments, synthetic nanocarriers are flat orplate-shaped. In some embodiments, synthetic nanocarriers are cubes orcubic. In some embodiments, synthetic nanocarriers are ovals orellipses. In some embodiments, synthetic nanocarriers are cylinders,cones, or pyramids.

In some embodiments, it is desirable to use a population of syntheticnanocarriers that is relatively uniform in terms of size or shape sothat each synthetic nanocarrier has similar properties. For example, atleast 80%, at least 90%, or at least 95% of the synthetic nanocarriers,based on the total number of synthetic nanocarriers, may have a minimumdimension or maximum dimension that falls within 5%, 10%, or 20% of theaverage diameter or average dimension of the synthetic nanocarriers.

Synthetic nanocarriers can be solid or hollow and can comprise one ormore layers. In some embodiments, each layer has a unique compositionand unique properties relative to the other layer(s). To give but oneexample, synthetic nanocarriers may have a core/shell structure, whereinthe core is one layer (e.g. a polymeric core) and the shell is a secondlayer (e.g. a lipid bilayer or monolayer). Synthetic nanocarriers maycomprise a plurality of different layers.

In some embodiments, synthetic nanocarriers may optionally comprise oneor more lipids. In some embodiments, a synthetic nanocarrier maycomprise a liposome. In some embodiments, a synthetic nanocarrier maycomprise a lipid bilayer. In some embodiments, a synthetic nanocarriermay comprise a lipid monolayer. In some embodiments, a syntheticnanocarrier may comprise a micelle. In some embodiments, a syntheticnanocarrier may comprise a core comprising a polymeric matrix surroundedby a lipid layer (e.g., lipid bilayer, lipid monolayer, etc.). In someembodiments, a synthetic nanocarrier may comprise a non-polymeric core(e.g., metal particle, quantum dot, ceramic particle, bone particle,viral particle, proteins, nucleic acids, carbohydrates, etc.) surroundedby a lipid layer (e.g., lipid bilayer, lipid monolayer, etc.).

In other embodiments, synthetic nanocarriers may comprise metalparticles, quantum dots, ceramic particles, etc. In some embodiments, anon-polymeric synthetic nanocarrier is an aggregate of non-polymericcomponents, such as an aggregate of metal atoms (e.g., gold atoms).

In some embodiments, synthetic nanocarriers may optionally comprise oneor more amphiphilic entities. In some embodiments, an amphiphilic entitycan promote the production of synthetic nanocarriers with increasedstability, improved uniformity, or increased viscosity. In someembodiments, amphiphilic entities can be associated with the interiorsurface of a lipid membrane (e.g., lipid bilayer, lipid monolayer,etc.). Many amphiphilic entities known in the art are suitable for usein making synthetic nanocarriers in accordance with the presentinvention. Such amphiphilic entities include, but are not limited to,phosphoglycerides; phosphatidylcholines; dipalmitoyl phosphatidylcholine(DPPC); dioleylphosphatidyl ethanolamine (DOPE);dioleyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine;cholesterol; cholesterol ester; diacylglycerol; diacylglycerolsuccinate;diphosphatidyl glycerol (DPPG); hexanedecanol; fatty alcohols such aspolyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; a surfaceactive fatty acid, such as palmitic acid or oleic acid; fatty acids;fatty acid monoglycerides; fatty acid diglycerides; fatty acid amides;sorbitan trioleate (Span®85) glycocholate; sorbitan monolaurate(Span®20); polysorbate 20 (Tween®20); polysorbate 60 (Tween®60);polysorbate 65 (Tween®65); polysorbate 80 (Tween®80); polysorbate 85(Tween®85); polyoxyethylene monostearate; surfactin; a poloxomer; asorbitan fatty acid ester such as sorbitan trioleate; lecithin;lysolecithin; phosphatidylserine; phosphatidylinositol; sphingomyelin;phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic acid;cerebrosides; dicetylphosphate; dipalmitoylphosphatidylglycerol;stearylamine; dodecylamine; hexadecyl-amine; acetyl palmitate; glycerolricinoleate; hexadecyl sterate; isopropyl myristate; tyloxapol;poly(ethylene glycol)5000-phosphatidylethanolamine; poly(ethyleneglycol)400-monostearate; phospholipids; synthetic and/or naturaldetergents having high surfactant properties; deoxycholates;cyclodextrins; chaotropic salts; ion pairing agents; and combinationsthereof. An amphiphilic entity component may be a mixture of differentamphiphilic entities. Those skilled in the art will recognize that thisis an exemplary, not comprehensive, list of substances with surfactantactivity. Any amphiphilic entity may be used in the production ofsynthetic nanocarriers to be used in accordance with the presentinvention.

In some embodiments, synthetic nanocarriers may optionally comprise oneor more carbohydrates. Carbohydrates may be natural or synthetic. Acarbohydrate may be a derivatized natural carbohydrate. In certainembodiments, a carbohydrate comprises monosaccharide or disaccharide,including but not limited to glucose, fructose, galactose, ribose,lactose, sucrose, maltose, trehalose, cellbiose, mannose, xylose,arabinose, glucoronic acid, galactoronic acid, mannuronic acid,glucosamine, galatosamine, and neuramic acid. In certain embodiments, acarbohydrate is a polysaccharide, including but not limited to pullulan,cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose(HPMC), hydroxycellulose (HC), methylcellulose (MC), dextran,cyclodextran, glycogen, hydroxyethylstarch, carageenan, glycon, amylose,chitosan, N,O-carboxylmethylchitosan, algin and alginic acid, starch,chitin, inulin, konjac, glucommannan, pustulan, heparin, hyaluronicacid, curdlan, and xanthan. In embodiments, the synthetic nanocarriersdo not comprise (or specifically exclude) carbohydrates, such as apolysaccharide. In certain embodiments, the carbohydrate may comprise acarbohydrate derivative such as a sugar alcohol, including but notlimited to mannitol, sorbitol, xylitol, erythritol, maltitol, andlactitol.

In some embodiments, synthetic nanocarriers can comprise one or morepolymers. In some embodiments, the synthetic nanocarriers comprise oneor more polymers that is a non-methoxy-terminated, pluronic polymer. Insome embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or99% (weight/weight) of the polymers that make up the syntheticnanocarriers are non-methoxy-terminated, pluronic polymers. In someembodiments, all of the polymers that make up the synthetic nanocarriersare non-methoxy-terminated, pluronic polymers. In some embodiments, thesynthetic nanocarriers comprise one or more polymers that is anon-methoxy-terminated polymer. In some embodiments, at least 1%, 2%,3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (weight/weight) of thepolymers that make up the synthetic nanocarriers arenon-methoxy-terminated polymers. In some embodiments, all of thepolymers that make up the synthetic nanocarriers arenon-methoxy-terminated polymers. In some embodiments, the syntheticnanocarriers comprise one or more polymers that do not comprise pluronicpolymer. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 97%, or 99% (weight/weight) of the polymers that make up thesynthetic nanocarriers do not comprise pluronic polymer. In someembodiments, all of the polymers that make up the synthetic nanocarriersdo not comprise pluronic polymer. In some embodiments, such a polymercan be surrounded by a coating layer (e.g., liposome, lipid monolayer,micelle, etc.). In some embodiments, various elements of the syntheticnanocarriers can be attached to the polymer.

The immunosuppressants can be attached to the synthetic nanocarriers byany of a number of methods. Generally, the attaching can be a result ofbonding between the immunosuppressants and the synthetic nanocarriers.This bonding can result in the immunosuppressants being attached to thesurface of the synthetic nanocarriers and/or contained (encapsulated)within the synthetic nanocarriers. In some embodiments, however, theimmunosuppressants are encapsulated by the synthetic nanocarriers as aresult of the structure of the synthetic nanocarriers rather thanbonding to the synthetic nanocarriers. In preferable embodiments, thesynthetic nanocarrier comprises a polymer as provided herein, and theimmunosuppressants are attached to the polymer.

When attaching occurs as a result of bonding between theimmunosuppressants and synthetic nanocarriers, the attaching may occurvia a coupling moiety. A coupling moiety can be any moiety through whichan immunosuppressant is bonded to a synthetic nanocarrier. Such moietiesinclude covalent bonds, such as an amide bond or ester bond, as well asseparate molecules that bond (covalently or non-covalently) theimmunosuppressant to the synthetic nanocarrier. Such molecules includelinkers or polymers or a unit thereof. For example, the coupling moietycan comprise a charged polymer to which an immunosuppressantelectrostatically binds. As another example, the coupling moiety cancomprise a polymer or unit thereof to which it is covalently bonded.

In preferred embodiments, the synthetic nanocarriers comprise a polymeras provided herein. These synthetic nanocarriers can be completelypolymeric or they can be a mix of polymers and other materials.

In some embodiments, the polymers of a synthetic nanocarrier associateto form a polymeric matrix. In some of these embodiments, a component,such as an immunosuppressant, can be covalently associated with one ormore polymers of the polymeric matrix. In some embodiments, covalentassociation is mediated by a linker. In some embodiments, a componentcan be noncovalently associated with one or more polymers of thepolymeric matrix. For example, in some embodiments, a component can beencapsulated within, surrounded by, and/or dispersed throughout apolymeric matrix. Alternatively or additionally, a component can beassociated with one or more polymers of a polymeric matrix byhydrophobic interactions, charge interactions, van der Waals forces,etc. A wide variety of polymers and methods for forming polymericmatrices therefrom are known conventionally.

Polymers may be natural or unnatural (synthetic) polymers. Polymers maybe homopolymers or copolymers comprising two or more monomers. In termsof sequence, copolymers may be random, block, or comprise a combinationof random and block sequences. Typically, polymers in accordance withthe present invention are organic polymers.

In some embodiments, the polymer comprises a polyester, polycarbonate,polyamide, or polyether, or unit thereof. In other embodiments, thepolymer comprises poly(ethylene glycol) (PEG), polypropylene glycol,poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid),or a polycaprolactone, or unit thereof. In some embodiments, it ispreferred that the polymer is biodegradable. Therefore, in theseembodiments, it is preferred that if the polymer comprises a polyether,such as poly(ethylene glycol) or polypropylene glycol or unit thereof,the polymer comprises a block-co-polymer of a polyether and abiodegradable polymer such that the polymer is biodegradable. In otherembodiments, the polymer does not solely comprise a polyether or unitthereof, such as poly(ethylene glycol) or polypropylene glycol or unitthereof.

Other examples of polymers suitable for use in the present inventioninclude, but are not limited to polyethylenes, polycarbonates (e.g.poly(1,3-dioxan-2one)), polyanhydrides (e.g. poly(sebacic anhydride)),polypropylfumerates, polyamides (e.g. polycaprolactam), polyacetals,polyethers, polyesters (e.g., polylactide, polyglycolide,polylactide-co-glycolide, polycaprolactone, polyhydroxyacid (e.g.poly(β-hydroxyalkanoate))), poly(orthoesters), polycyanoacrylates,polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polyureas, polystyrenes, and polyamines, polylysine,polylysine-PEG copolymers, and poly(ethyleneimine), poly(ethyleneimine)-PEG copolymers.

In some embodiments, polymers in accordance with the present inventioninclude polymers which have been approved for use in humans by the U.S.Food and Drug Administration (FDA) under 21 C.F.R. § 177.2600, includingbut not limited to polyesters (e.g., polylactic acid,poly(lactic-co-glycolic acid), polycaprolactone, polyvalerolactone,poly(1,3-dioxan-2one)); polyanhydrides (e.g., poly(sebacic anhydride));polyethers (e.g., polyethylene glycol); polyurethanes;polymethacrylates; polyacrylates; and polycyanoacrylates.

In some embodiments, polymers can be hydrophilic. For example, polymersmay comprise anionic groups (e.g., phosphate group, sulphate group,carboxylate group); cationic groups (e.g., quaternary amine group); orpolar groups (e.g., hydroxyl group, thiol group, amine group). In someembodiments, a synthetic nanocarrier comprising a hydrophilic polymericmatrix generates a hydrophilic environment within the syntheticnanocarrier. In some embodiments, polymers can be hydrophobic. In someembodiments, a synthetic nanocarrier comprising a hydrophobic polymericmatrix generates a hydrophobic environment within the syntheticnanocarrier. Selection of the hydrophilicity or hydrophobicity of thepolymer may have an impact on the nature of materials that areincorporated (e.g. attached) within the synthetic nanocarrier.

In some embodiments, polymers may be modified with one or more moietiesand/or functional groups. A variety of moieties or functional groups canbe used in accordance with the present invention. In some embodiments,polymers may be modified with polyethylene glycol (PEG), with acarbohydrate, and/or with acyclic polyacetals derived frompolysaccharides (Papisov, 2001, ACS Symposium Series, 786:301). Certainembodiments may be made using the general teachings of U.S. Pat. No.5,543,158 to Gref et al., or WO publication WO2009/051837 by Von Andrianet al.

In some embodiments, polymers may be modified with a lipid or fatty acidgroup. In some embodiments, a fatty acid group may be one or more ofbutyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic,arachidic, behenic, or lignoceric acid. In some embodiments, a fattyacid group may be one or more of palmitoleic, oleic, vaccenic, linoleic,alpha-linoleic, gamma-linoleic, arachidonic, gadoleic, arachidonic,eicosapentaenoic, docosahexaenoic, or erucic acid.

In some embodiments, polymers may be polyesters, including copolymerscomprising lactic acid and glycolic acid units, such as poly(lacticacid-co-glycolic acid) and poly(lactide-co-glycolide), collectivelyreferred to herein as “PLGA”; and homopolymers comprising glycolic acidunits, referred to herein as “PGA,” and lactic acid units, such aspoly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid,poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectivelyreferred to herein as “PLA.” In some embodiments, exemplary polyestersinclude, for example, polyhydroxyacids; PEG copolymers and copolymers oflactide and glycolide (e.g., PLA-PEG copolymers, PGA-PEG copolymers,PLGA-PEG copolymers, and derivatives thereof. In some embodiments,polyesters include, for example, poly(caprolactone),poly(caprolactone)-PEG copolymers, poly(L-lactide-co-L-lysine),poly(serine ester), poly(4-hydroxy-L-proline ester),poly[α-(4-aminobutyl)-L-glycolic acid], and derivatives thereof.

In some embodiments, a polymer may be PLGA. PLGA is a biocompatible andbiodegradable co-polymer of lactic acid and glycolic acid, and variousforms of PLGA are characterized by the ratio of lactic acid:glycolicacid. Lactic acid can be L-lactic acid, D-lactic acid, or D,L-lacticacid. The degradation rate of PLGA can be adjusted by altering thelactic acid:glycolic acid ratio. In some embodiments, PLGA to be used inaccordance with the present invention is characterized by a lacticacid:glycolic acid ratio of approximately 85:15, approximately 75:25,approximately 60:40, approximately 50:50, approximately 40:60,approximately 25:75, or approximately 15:85.

In some embodiments, polymers may be one or more acrylic polymers. Incertain embodiments, acrylic polymers include, for example, acrylic acidand methacrylic acid copolymers, methyl methacrylate copolymers,ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkylmethacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),methacrylic acid alkylamide copolymer, poly(methyl methacrylate),poly(methacrylic acid anhydride), methyl methacrylate, polymethacrylate,poly(methyl methacrylate) copolymer, polyacrylamide, aminoalkylmethacrylate copolymer, glycidyl methacrylate copolymers,polycyanoacrylates, and combinations comprising one or more of theforegoing polymers. The acrylic polymer may comprise fully-polymerizedcopolymers of acrylic and methacrylic acid esters with a low content ofquaternary ammonium groups.

In some embodiments, polymers can be cationic polymers. In general,cationic polymers are able to condense and/or protect negatively chargedstrands of nucleic acids (Zauner et al., 1998, Adv. Drug Del. Rev.,30:97; and Kabanov et al., 1995, Bioconjugate Chem., 6:7), poly(ethyleneimine) (PEI; Boussif et al., 1995, Proc. Natl. Acad. Sci., USA, 1995,92:7297), and poly(amidoamine) dendrimers (Kukowska-Latallo et al.,1996, Proc. Natl. Acad. Sci., USA, 93:4897; Tang et al., 1996,Bioconjugate Chem., 7:703; and Haensler et al., 1993, BioconjugateChem., 4:372) are positively-charged at physiological pH, form ion pairswith nucleic acids. In embodiments, the synthetic nanocarriers may notcomprise (or may exclude) cationic polymers.

In some embodiments, polymers can be degradable polyesters bearingcationic side chains (Putnam et al., 1999, Macromolecules, 32:3658;Barrera et al., 1993, J. Am. Chem. Soc., 115:11010; Kwon et al., 1989,Macromolecules, 22:3250; Lim et al., 1999, J. Am. Chem. Soc., 121:5633;and Zhou et al., 1990, Macromolecules, 23:3399). Examples of thesepolyesters include poly(L-lactide-co-L-lysine) (Barrera et al., 1993, J.Am. Chem. Soc., 115:11010), poly(serine ester) (Zhou et al., 1990,Macromolecules, 23:3399), poly(4-hydroxy-L-proline ester) (Putnam etal., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem.Soc., 121:5633), and poly(4-hydroxy-L-proline ester) (Putnam et al.,1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem. Soc.,121:5633).

The properties of these and other polymers and methods for preparingthem are well known in the art (see, for example, U.S. Pat. Nos.6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404; 6,095,148;5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600; 5,399,665;5,019,379; 5,010,167; 4,806,621; 4,638,045; and U.S. Pat. No. 4,946,929;Wang et al., 2001, J. Am. Chem. Soc., 123:9480; Lim et al., 2001, J. Am.Chem. Soc., 123:2460; Langer, 2000, Acc. Chem. Res., 33:94; Langer,1999, J. Control. Release, 62:7; and Uhrich et al., 1999, Chem. Rev.,99:3181). More generally, a variety of methods for synthesizing certainsuitable polymers are described in Concise Encyclopedia of PolymerScience and Polymeric Amines and Ammonium Salts, Ed. by Goethals,Pergamon Press, 1980; Principles of Polymerization by Odian, John Wiley& Sons, Fourth Edition, 2004; Contemporary Polymer Chemistry by Allcocket al., Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; andin U.S. Pat. Nos. 6,506,577, 6,632,922, 6,686,446, and 6,818,732.

In some embodiments, polymers can be linear or branched polymers. Insome embodiments, polymers can be dendrimers. In some embodiments,polymers can be substantially cross-linked to one another. In someembodiments, polymers can be substantially free of cross-links. In someembodiments, polymers can be used in accordance with the presentinvention without undergoing a cross-linking step. It is further to beunderstood that the synthetic nanocarriers may comprise blockcopolymers, graft copolymers, blends, mixtures, and/or adducts of any ofthe foregoing and other polymers. Those skilled in the art willrecognize that the polymers listed herein represent an exemplary, notcomprehensive, list of polymers that can be of use in accordance withthe present invention.

In some embodiments, synthetic nanocarriers do not comprise a polymericcomponent. In some embodiments, synthetic nanocarriers may comprisemetal particles, quantum dots, ceramic particles, etc. In someembodiments, a non-polymeric synthetic nanocarrier is an aggregate ofnon-polymeric components, such as an aggregate of metal atoms (e.g.,gold atoms).

Compositions according to the invention can comprise elements, such asimmunosuppressants, in combination with pharmaceutically acceptableexcipients, such as preservatives, buffers, saline, or phosphatebuffered saline. The compositions may be made using conventionalpharmaceutical manufacturing and compounding techniques to arrive atuseful dosage forms. In an embodiment, compositions, such as thosecomprising synthetic nanocarriers, are suspended in sterile salinesolution for injection together with a preservative.

In embodiments, when preparing synthetic nanocarriers as carriers,methods for attaching components to the synthetic nanocarriers may beuseful. If the component is a small molecule it may be of advantage toattach the component to a polymer prior to the assembly of the syntheticnanocarriers. In embodiments, it may also be an advantage to prepare thesynthetic nanocarriers with surface groups that are used to attach thecomponent to the synthetic nanocarrier through the use of these surfacegroups rather than attaching the component to a polymer and then usingthis polymer conjugate in the construction of synthetic nanocarriers.

In certain embodiments, the attaching can be with a covalent linker. Inembodiments, immunosuppressants according to the invention can becovalently attached to the external surface via a 1,2,3-triazole linkerformed by the 1,3-dipolar cycloaddition reaction of azido groups on thesurface of the nanocarrier with immunosuppressant containing an alkynegroup or by the 1,3-dipolar cycloaddition reaction of alkynes on thesurface of the nanocarrier with immunosuppressants containing an azidogroup. Such cycloaddition reactions are preferably performed in thepresence of a Cu(I) catalyst along with a suitable Cu(I)-ligand and areducing agent to reduce Cu(II) compound to catalytic active Cu(I)compound. This Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) canalso be referred as the click reaction.

Additionally, the covalent coupling may comprise a covalent linker thatcomprises an amide linker, a disulfide linker, a thioether linker, ahydrazone linker, a hydrazide linker, an imine or oxime linker, an ureaor thiourea linker, an amidine linker, an amine linker, and asulfonamide linker.

An amide linker is formed via an amide bond between an amine on onecomponent such as an immunosuppressant with the carboxylic acid group ofa second component such as the nanocarrier. The amide bond in the linkercan be made using any of the conventional amide bond forming reactionswith suitably protected amino acids and activated carboxylic acid suchN-hydroxy succinimide-activated ester.

A disulfide linker is made via the formation of a disulfide (S—S) bondbetween two sulfur atoms of the form, for instance, of R1-S—S—R2. Adisulfide bond can be formed by thiol exchange of a component containingthiol/mercaptan group (—SH) with another activated thiol group on apolymer or nanocarrier or a nanocarrier containing thiol/mercaptangroups with a component containing activated thiol group.

A triazole linker, specifically a 1,2,3-triazole of the form

wherein R1 and R2 may be any chemical entities, is made by the1,3-dipolar cycloaddition reaction of an azide attached to a firstcomponent such as the nanocarrier with a terminal alkyne attached to asecond component such as the immunosuppressant. The 1,3-dipolarcycloaddition reaction is performed with or without a catalyst,preferably with Cu(I)-catalyst, which links the two components through a1,2,3-triazole function. This chemistry is described in detail bySharpless et al., Angew. Chem. Int. Ed. 41(14), 2596, (2002) and Meldal,et al, Chem. Rev., 2008, 108(8), 2952-3015 and is often referred to as a“click” reaction or CuAAC.

In embodiments, a polymer containing an azide or alkyne group, terminalto the polymer chain is prepared. This polymer is then used to prepare asynthetic nanocarrier in such a manner that a plurality of the alkyne orazide groups are positioned on the surface of that nanocarrier.Alternatively, the synthetic nanocarrier can be prepared by anotherroute, and subsequently functionalized with alkyne or azide groups. Thecomponent is prepared with the presence of either an alkyne (if thepolymer contains an azide) or an azide (if the polymer contains analkyne) group. The component is then allowed to react with thenanocarrier via the 1,3-dipolar cycloaddition reaction with or without acatalyst which covalently attaches the component to the particle throughthe 1,4-disubstituted 1,2,3-triazole linker.

A thioether linker is made by the formation of a sulfur-carbon(thioether) bond in the form, for instance, of R1-S—R2. Thioether can bemade by either alkylation of a thiol/mercaptan (—SH) group on onecomponent with an alkylating group such as halide or epoxide on a secondcomponent. Thioether linkers can also be formed by Michael addition of athiol/mercaptan group on one component to an electron-deficient alkenegroup on a second component containing a maleimide group or vinylsulfone group as the Michael acceptor. In another way, thioether linkerscan be prepared by the radical thiol-ene reaction of a thiol/mercaptangroup on one component with an alkene group on a second component.

A hydrazone linker is made by the reaction of a hydrazide group on onecomponent with an aldehyde/ketone group on the second component.

A hydrazide linker is formed by the reaction of a hydrazine group on onecomponent with a carboxylic acid group on the second component. Suchreaction is generally performed using chemistry similar to the formationof amide bond where the carboxylic acid is activated with an activatingreagent.

An imine or oxime linker is formed by the reaction of an amine orN-alkoxyamine (or aminooxy) group on one component with an aldehyde orketone group on the second component.

An urea or thiourea linker is prepared by the reaction of an amine groupon one component with an isocyanate or thioisocyanate group on thesecond component.

An amidine linker is prepared by the reaction of an amine group on onecomponent with an imidoester group on the second component.

An amine linker is made by the alkylation reaction of an amine group onone component with an alkylating group such as halide, epoxide, orsulfonate ester group on the second component. Alternatively, an aminelinker can also be made by reductive amination of an amine group on onecomponent with an aldehyde or ketone group on the second component witha suitable reducing reagent such as sodium cyanoborohydride or sodiumtriacetoxyborohydride.

A sulfonamide linker is made by the reaction of an amine group on onecomponent with a sulfonyl halide (such as sulfonyl chloride) group onthe second component.

A sulfone linker is made by Michael addition of a nucleophile to a vinylsulfone. Either the vinyl sulfone or the nucleophile may be on thesurface of the nanocarrier or attached to a component.

The component can also be conjugated to the nanocarrier via non-covalentconjugation methods. For example, a negative charged immunosuppressantcan be conjugated to a positive charged nanocarrier throughelectrostatic adsorption. A component containing a metal ligand can alsobe conjugated to a nanocarrier containing a metal complex via ametal-ligand complex.

In embodiments, the component can be attached to a polymer, for examplepolylactic acid-block-polyethylene glycol, prior to the assembly of thesynthetic nanocarrier or the synthetic nanocarrier can be formed withreactive or activatible groups on its surface. In the latter case, thecomponent may be prepared with a group which is compatible with theattachment chemistry that is presented by the synthetic nanocarriers'surface. In other embodiments, a peptide component can be attached toVLPs or liposomes using a suitable linker. A linker is a compound orreagent that is capable of coupling two molecules together. In anembodiment, the linker can be a homobifuntional or heterobifunctionalreagent as described in Hermanson 2008. For example, an VLP or liposomesynthetic nanocarrier containing a carboxylic group on the surface canbe treated with a homobifunctional linker, adipic dihydrazide (ADH), inthe presence of EDC to form the corresponding synthetic nanocarrier withthe ADH linker. The resulting ADH linked synthetic nanocarrier is thenconjugated with a peptide component containing an acid group via theother end of the ADH linker on nanocarrier to produce the correspondingVLP or liposome peptide conjugate.

For detailed descriptions of available conjugation methods, seeHermanson G T “Bioconjugate Techniques”, 2nd Edition Published byAcademic Press, Inc., 2008. In addition to covalent attachment thecomponent can be attached by adsorption to a pre-formed syntheticnanocarrier or it can be attached by encapsulation during the formationof the synthetic nanocarrier.

Any immunosuppressant as provided herein can be used in the methods orcompositions provided and can be, in some embodiments, attached tosynthetic nanocarriers. Immunosuppressants include, but are not limitedto, statins; mTOR inhibitors, such as rapamycin or a rapamycin analog;TGF-β signaling agents; TGF-β receptor agonists; histone deacetylase(HDAC) inhibitors; corticosteroids; inhibitors of mitochondrialfunction, such as rotenone; P38 inhibitors; NF-κβ inhibitors; adenosinereceptor agonists; prostaglandin E2 agonists; phosphodiesteraseinhibitors, such as phosphodiesterase 4 inhibitor; proteasomeinhibitors; kinase inhibitors; G-protein coupled receptor agonists;G-protein coupled receptor antagonists; glucocorticoids; retinoids;cytokine inhibitors; cytokine receptor inhibitors; cytokine receptoractivators; peroxisome proliferator-activated receptor antagonists;peroxisome proliferator-activated receptor agonists; histone deacetylaseinhibitors; calcineurin inhibitors; phosphatase inhibitors and oxidizedATPs. Immunosuppressants also include IDO, vitamin D3, cyclosporine A,aryl hydrocarbon receptor inhibitors, resveratrol, azathiopurine,6-mercaptopurine, aspirin, niflumic acid, estriol, tripolide,interleukins (e.g., IL-1, IL-10), cyclosporine A, siRNAs targetingcytokines or cytokine receptors and the like.

Examples of statins include atorvastatin (LIPITOR®, TORVAST®),cerivastatin, fluvastatin (LESCOL®, LESCOL® XL), lovastatin (MEVACOR®,ALTOCOR®, ALTOPREV®), mevastatin (COMPACTIN®), pitavastatin (LIVALO®,PIAVA®), rosuvastatin (PRAVACHOL®, SELEKTINE®, LIPOSTAT®), rosuvastatin(CRESTOR®), and simvastatin (ZOCOR®, LIPEX®).

Examples of mTOR inhibitors include rapamycin and analogs thereof (e.g.,CCL-779, RAD001, AP23573, C20-methallylrapamycin (C20-Marap),C16-(S)-butylsulfonamidorapamycin (C16-BSrap),C16-(S)-3-methylindolerapamycin (C16-iRap) (Bayle et al. Chemistry &Biology 2006, 13:99-107)), AZD8055, BEZ235 (NVP-BEZ235), chrysophanicacid (chrysophanol), deforolimus (MK-8669), everolimus (RAD0001),KU-0063794, PI-103, PP242, temsirolimus, and WYE-354 (available fromSelleck, Houston, Tex., USA).

Examples of TGF-β signaling agents include TGF-β ligands (e.g., activinA, GDF1, GDF11, bone morphogenic proteins, nodal, TGF-βs) and theirreceptors (e.g., ACVR1B, ACVR1C, ACVR2A, ACVR2B, BMPR2, BMPR1A, BMPR1B,TGFβRI, TGFβRII), R-SMADS/co-SMADS (e.g., SMAD1, SMAD2, SMAD3, SMAD4,SMAD5, SMAD8), and ligand inhibitors (e.g, follistatin, noggin, chordin,DAN, lefty, LTBP1, THBS1, Decorin).

Examples of inhibitors of mitochondrial function include atractyloside(dipotassium salt), bongkrekic acid (triammonium salt), carbonyl cyanidem-chlorophenylhydrazone, carboxyatractyloside (e.g., from Atractylisgummifera), CGP-37157, (−)-Deguelin (e.g., from Mundulea sericea), F16,hexokinase II VDAC binding domain peptide, oligomycin, rotenone, Ru360,SFK1, and valinomycin (e.g., from Streptomyces fulvissimus)(EMD4Biosciences, USA).

Examples of P38 inhibitors include SB-203580(4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole),SB-239063(trans-1-(4hydroxycyclohexyl)-4-(fluorophenyl)-5-(2-methoxy-pyrimidin-4-yl)imidazole), SB-220025(5-(2amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(4-piperidinyl)imidazole)),and ARRY-797.

Examples of NF (e.g., NK-κβ) inhibitors include IFRD1,2-(1,8-naphthyridin-2-yl)-Phenol, 5-aminosalicylic acid, BAY 11-7082,BAY 11-7085, CAPE (Caffeic Acid Phenethylester), diethylmaleate, IKK-2Inhibitor IV, IMD 0354, lactacystin, MG-132 [Z-Leu-Leu-Leu-CHO], NFκBActivation Inhibitor III, NF-κB Activation Inhibitor II, JSH-23,parthenolide, Phenylarsine Oxide (PAO), PPM-18,pyrrolidinedithiocarbamic acid ammonium salt, QNZ, RO 106-9920,rocaglamide, rocaglamide AL, rocaglamide C, rocaglamide I, rocaglamideJ, rocaglaol, (R)-MG-132, sodium salicylate, triptolide (PG490), andwedelolactone.

Examples of adenosine receptor agonists include CGS-21680 and ATL-146e.

Examples of prostaglandin E2 agonists include E-Prostanoid 2 andE-Prostanoid 4.

Examples of phosphodiesterase inhibitors (non-selective and selectiveinhibitors) include caffeine, aminophylline, IBMX(3-isobutyl-1-methylxanthine), paraxanthine, pentoxifylline,theobromine, theophylline, methylated xanthines, vinpocetine, EHNA(erythro-9-(2-hydroxy-3-nonyl)adenine), anagrelide, enoximone (PERFAN™),milrinone, levosimendon, mesembrine, ibudilast, piclamilast, luteolin,drotaverine, roflumilast (DAXAS™, DALIRESP™), sildenafil (REVATION®,VIAGRA®), tadalafil (ADCIRCA®, CIALIS®), vardenafil (LEVITRA®, STAXYN®),udenafil, avanafil, icariin, 4-methylpiperazine, and pyrazolopyrimidin-7-1.

Examples of proteasome inhibitors include bortezomib, disulfiram,epigallocatechin-3-gallate, and salinosporamide A.

Examples of kinase inhibitors include bevacizumab, BIBW 2992, cetuximab(ERBITUX®), imatinib (GLEEVEC®), trastuzumab (HERCEPTIN®), gefitinib(IRESSA®), ranibizumab (LUCENTIS®), pegaptanib, sorafenib, dasatinib,sunitinib, erlotinib, nilotinib, lapatinib, panitumumab, vandetanib,E7080, pazopanib, and mubritinib.

Examples of glucocorticoids include hydrocortisone (cortisol), cortisoneacetate, prednisone, prednisolone, methylprednisolone, dexamethasone,betamethasone, triamcinolone, beclometasone, fludrocortisone acetate,deoxycorticosterone acetate (DOCA), and aldosterone.

Examples of retinoids include retinol, retinal, tretinoin (retinoicacid, RETIN-A®), isotretinoin (ACCUTANE®, AMNESTEEM®, CLARAVIS®,SOTRET®), alitretinoin (PANRETIN®), etretinate (TEGISON™) and itsmetabolite acitretin (SORIATANE®), tazarotene (TAZORAC®, AVAGE®,ZORAC®), bexarotene (TARGRETIN®), and adapalene (DIFFERIN®).

Examples of cytokine inhibitors include IL1ra, IL1 receptor antagonist,IGFBP, TNF-BF, uromodulin, Alpha-2-Macroglobulin, Cyclosporin A,Pentamidine, and Pentoxifylline (PENTOPAK®, PENTOXIL®, TRENTAL®).

Examples of peroxisome proliferator-activated receptor antagonistsinclude GW9662, PPARγ antagonist III, G335, and T0070907(EMD4Biosciences, USA).

Examples of peroxisome proliferator-activated receptor agonists includepioglitazone, ciglitazone, clofibrate, GW1929, GW7647, L-165,041, LY171883, PPARγ activator, Fmoc-Leu, troglitazone, and WY-14643(EMD4Biosciences, USA).

Examples of histone deacetylase inhibitors include hydroxamic acids (orhydroxamates) such as trichostatin A, cyclic tetrapeptides (such astrapoxin B) and depsipeptides, benzamides, electrophilic ketones,aliphatic acid compounds such as phenylbutyrate and valproic acid,hydroxamic acids such as vorinostat (SAHA), belinostat (PXD101), LAQ824,and panobinostat (LBH589), benzamides such as entinostat (MS-275),CI994, and mocetinostat (MGCD0103), nicotinamide, derivatives of NAD,dihydrocoumarin, naphthopyranone, and 2-hydroxynaphaldehydes.

Examples of calcineurin inhibitors include cyclosporine, pimecrolimus,voclosporin, and tacrolimus.

Examples of phosphatase inhibitors include BN82002 hydrochloride,CP-91149, calyculin A, cantharidic acid, cantharidin, cypermethrin,ethyl-3,4-dephostatin, fostriecin sodium salt, MAZ51,methyl-3,4-dephostatin, NSC 95397, norcantharidin, okadaic acid ammoniumsalt from prorocentrum concavum, okadaic acid, okadaic acid potassiumsalt, okadaic acid sodium salt, phenylarsine oxide, various phosphataseinhibitor cocktails, protein phosphatase 1C, protein phosphatase 2Ainhibitor protein, protein phosphatase 2A1, protein phosphatase 2A2, andsodium orthovanadate.

In some embodiments, therapeutic macromolecules may be delivered in theform of the therapeutic macromolecule itself, or fragments orderivatives thereof. Therapeutic macromolecules can include therapeuticproteins or therapeutic polynucleotides.

Therapeutic proteins include, but are not limited to, infusibletherapeutic proteins, enzymes, enzyme cofactors, hormones, bloodclotting factors, cytokines and interferons, growth factors, monoclonalantibodies, and polyclonal antibodies (e.g., that are administered to asubject as a replacement therapy), and proteins associated with Pompe'sdisease (e.g., acid glucosidase alfa, rhGAA (e.g., Myozyme and Lumizyme(Genzyme)). Therapeutic proteins also include proteins involved in theblood coagulation cascade. Therapeutic proteins include, but are notlimited to, Factor VIII, Factor VII, Factor IX, Factor V, von WillebrandFactor, von Heldebrant Factor, tissue plasminogen activator, insulin,growth hormone, erythropoietin alfa, VEGF, thrombopoietin, lysozyme,antithrombin and the like. Therapeutic proteins also include adipokines,such as leptin and adiponectin. Other examples of therapeutic proteinsare as described below and elsewhere herein.

Examples of therapeutic proteins used in enzyme replacement therapy ofsubjects having a lysosomal storage disorder include, but are notlimited to, imiglucerase for the treatment of Gaucher's disease (e.g.,CEREZYME™), a-galactosidase A (a-gal A) for the treatment of Fabrydisease (e.g., agalsidase beta, FABRYZYME™), acid α-glucosidase (GAA)for the treatment of Pompe disease (e.g., acid glucosidase alfa,LUMIZYME™, MYOZYME™), arylsulfatase B for the treatment ofMucopolysaccharidoses (e.g., laronidase, ALDURAZYME™, idursulfase,ELAPRASE™, arylsulfatase B, NAGLAZYME™), pegloticase (KRYSTEXXA) andpegsiticase.

Examples of enzymes include oxidoreductases, transferases, hydrolases,lyases, isomerases, asparaginases, uricases, glycosidases,asparaginases, uricases, proteases, nucleases, collagenases,hyaluronidases, heparinases, heparanases, lysins, and ligases.

Therapeutic proteins may also include any enzyme, toxin, or otherprotein or peptide isolated or derived from a bacterial, fungal, orviral source.

Examples of hormones include Melatonin (N-acetyl-5-methoxytryptamine),Serotonin, Thyroxine (or tetraiodothyronine) (a thyroid hormone),Triiodothyronine (a thyroid hormone), Epinephrine (or adrenaline),Norepinephrine (or noradrenaline), Dopamine (or prolactin inhibitinghormone), Antimullerian hormone (or mullerian inhibiting factor orhormone), Adiponectin, Adrenocorticotropic hormone (or corticotropin),Angiotensinogen and angiotensin, Antidiuretic hormone (or vasopressin,arginine vasopressin), Atrial-natriuretic peptide (or atriopeptin),Calcitonin, Cholecystokinin, Corticotropin-releasing hormone,Erythropoietin, Follicle-stimulating hormone, Gastrin, Ghrelin,Glucagon, Glucagon-like peptide (GLP-1), GIP, Gonadotropin-releasinghormone, Growth hormone-releasing hormone, Human chorionic gonadotropin,Human placental lactogen, Growth hormone, Inhibin, Insulin, Insulin-likegrowth factor (or somatomedin), Leptin, Luteinizing hormone, Melanocytestimulating hormone, Orexin, Oxytocin, Parathyroid hormone, Prolactin,Relaxin, Secretin, Somatostatin, Thrombopoietin, Thyroid-stimulatinghormone (or thyrotropin), Thyrotropin-releasing hormone, Cortisol,Aldosterone, Testosterone, Dehydroepiandrosterone, Androstenedione,Dihydrotestosterone, Estradiol, Estrone, Estriol, Progesterone,Calcitriol (1,25-dihydroxyvitamin D3), Calcidiol (25-hydroxyvitamin D3),Prostaglandins, Leukotrienes, Prostacyclin, Thromboxane, Prolactinreleasing hormone, Lipotropin, Brain natriuretic peptide, NeuropeptideY, Histamine, Endothelin, Pancreatic polypeptide, Renin, and Enkephalin.

Examples of blood or blood coagulation factors include Factor I(fibrinogen), Factor II (prothrombin), tissue factor, Factor V(proaccelerin, labile factor), Factor VII (stable factor, proconvertin),Factor VIII (antihemophilic globulin), Factor IX (Christmas factor orplasma thromboplastin component), Factor X (Stuart-Prower factor),Factor Xa, Factor XI, Factor XII (Hageman factor), Factor XIII(fibrin-stabilizing factor), von Willebrand factor, prekallikrein(Fletcher factor), high-molecular weight kininogen (HMWK) (Fitzgeraldfactor), fibronectin, fibrin, thrombin, antithrombin III, heparincofactor II, protein C, protein S, protein Z, protein Z-related proteaseinhibitor (ZPI), plasminogen, alpha 2-antiplasmin, tissue plasminogenactivator (tPA), urokinase, plasminogen activator inhibitor-1 (PAI1),plasminogen activator inhibitor-2 (PAI2), cancer procoagulant, andepoetin alfa (Epogen, Procrit).

Examples of cytokines include lymphokines, interleukins, and chemokines,type 1 cytokines, such as IFN-γ, TGF-β, and type 2 cytokines, such asIL-4, IL-10, and IL-13.

Examples of growth factors include Adrenomedullin (AM), Angiopoietin(Ang), Autocrine motility factor, Bone morphogenetic proteins (BMPs),Brain-derived neurotrophic factor (BDNF), Epidermal growth factor (EGF),Erythropoietin (EPO), Fibroblast growth factor (FGF), Glial cellline-derived neurotrophic factor (GDNF), Granulocyte colony-stimulatingfactor (G-CSF), Granulocyte macrophage colony-stimulating factor(GM-CSF), Growth differentiation factor-9 (GDF9), Hepatocyte growthfactor (HGF), Hepatoma-derived growth factor (HDGF), Insulin-like growthfactor (IGF), Migration-stimulating factor, Myostatin (GDF-8), Nervegrowth factor (NGF) and other neurotrophins, Platelet-derived growthfactor (PDGF), Thrombopoietin (TPO), Transforming growth factor alpha(TGF-α), Transforming growth factor beta (TGF-β), Tumour necrosisfactor-alpha (TNF-α), Vascular endothelial growth factor (VEGF), WntSignaling Pathway, placental growth factor (P1GF), (Foetal BovineSomatotrophin) (FBS), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, and IL-7.

Examples of monoclonal antibodies include Abagovomab, Abciximab,Adalimumab, Adecatumumab, Afelimomab, Afutuzumab, Alacizumab pegol, ALD,Alemtuzumab, Altumomab pentetate, Anatumomab mafenatox, Anrukinzumab,Anti-thymocyte globin, Apolizumab, Arcitumomab, Aselizumab, Atlizumab(tocilizumab), Atorolimumab, Bapineuzumab, Basiliximab, Bavituximab,Bectumomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab,Bevacizumab, Biciromab, Bivatuzumab mertansine, Blinatumomab,Brentuximab vedotin, Briakinumab, Canakinumab, Cantuzumab mertansine,Capromab pendetide, Catumaxomab, Cedelizumab, Certolizumab pegol,Cetuximab, Citatuzumab bogatox, Cixutumumab, Clenoliximab, Clivatuzumabtetraxetan, Conatumumab, Dacetuzumab, Daclizumab, Daratumumab,Denosumab, Detumomab, Dorlimomab aritox, Dorlixizumab, Ecromeximab,Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Elotuzumab,Elsilimomab, Enlimomab pegol, Epitumomab cituxetan, Epratuzumab,Erlizumab, Ertumaxomab, Etaracizumab, Exbivirumab, Fanolesomab,Faralimomab, Farletuzumab, Felvizumab, Fezakinumab, Figitumumab,Fontolizumab, Foravirumab, Fresolimumab, Galiximab, Gantenerumab,Gavilimomab, Gemtuzumab ozogamicin, GC1008, Girentuximab, Glembatumumabvedotin, Golimumab, Gomiliximab, Ibalizumab, Ibritumomab tiuxetan,Igovomab, Imciromab, Infliximab, Intetumumab, Inolimomab, Inotuzumabozogamicin, Ipilimumab, Iratumumab, Keliximab, Labetuzumab,Lebrikizumab, Lemalesomab, Lerdelimumab, Lexatumumab, Libivirumab,Lintuzumab, Lorvotuzumab mertansine, Lucatumumab, Lumiliximab,Mapatumumab, Maslimomab, Matuzumab, Mepolizumab, Metelimumab,Milatuzumab, Minretumomab, Mitumomab, Morolimumab, Motavizumab,Muromonab-CD3, Nacolomab tafenatox, Naptumomab estafenatox, Natalizumab,Nebacumab, Necitumumab, Nerelimomab, Nimotuzumab, Nofetumomab merpentan,Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Omalizumab, Oportuzumabmonatox, Oregovomab, Otelixizumab, Pagibaximab, Palivizumab,Panitumumab, Panobacumab, Pascolizumab, Pemtumomab, Pertuzumab,Pexelizumab, Pintumomab, Priliximab, Pritumumab, Rafivirumab,Ramucirumab, Ranibizumab, Raxibacumab, Regavirumab Reslizumab,Rilotumumab, Rituximab, Robatumumab, Rontalizumab, Rovelizumab,Ruplizumab, Satumomab pendetide, Sevirumab, Sibrotuzumab, Sifalimumab,Siltuximab, Siplizumab, Solanezumab, Sonepcizumab, Sontuzumab,Stamulumab, Sulesomab, Tacatuzumab tetraxetan, Tadocizumab, Talizumab,Tanezumab, Taplitumomab paptox, Tefibazumab, Telimomab aritox,Tenatumomab, Teneliximab, Teplizumab, Ticilimumab (tremelimumab),Tigatuzumab, Tocilizumab (atlizumab), Toralizumab, Tositumomab,Trastuzumab, Tremelimumab, Tucotuzumab celmoleukin, Tuvirumab,Urtoxazumab, Ustekinumab, Vapaliximab, Vedolizumab, Veltuzumab,Vepalimomab, Visilizumab, Volociximab, Votumumab, Zalutumumab,Zanolimumab, Ziralimumab, and Zolimomab aritox. Monoclonal antibodiesfurther include anti-TNF-□ antibodies.

Examples of infusion therapy or injectable therapeutic proteins include,for example, Tocilizumab (Roche/Actemra®), alpha-1 antitrypsin(Kamada/AAT), Hematide® (Affymax and Takeda, synthetic peptide),albinterferon alfa-2b (Novartis/Zalbin™), Rhucin® (Pharming Group, C1inhibitor replacement therapy), tesamorelin (Theratechnologies/Egrifta,synthetic growth hormone-releasing factor), ocrelizumab (Genentech,Roche and Biogen), belimumab (GlaxoSmithKline/Benlysta®), pegloticase(Savient Pharmaceuticals/Krystexxa™), pegsiticase, taliglucerase alfa(Protalix/Uplyso), agalsidase alfa (Shire/Replagal®), velaglucerase alfa(Shire), and Keyhole Limpet Hemocyanin (KLH).

Additional therapeutic proteins include, for example, engineeredproteins, such as Fc fusion proteins, bispecific antibodies,multi-specific antibodies, nanobodies, antigen-binding proteins,antibody fragments, and protein conjugates, such as antibody drugconjugates.

Therapeutic polynucleotides include, but are not limited to nucleic acidaptamers such as Pegaptanib (Macugen, a pegylated anti-VEGF aptamer),antisense therapeutics such as antisense poly- or oligonucleotides(e.g., antiviral drug Fomivirsen, or Mipomersen, an antisensetherapeutic that targets the messenger RNA for apolipoprotein B forreduction of cholesterol level); small interfering RNAs (siRNAs) (e.g.,dicer substrate siRNA molecules (DsiRNAs) which are 25-30 base pairasymmetric double-stranded RNAs that mediate RNAi with extremely highpotency); or modified messenger RNAs (mmRNAs) such as those disclosed inUS Patent application 2013/0115272 to de Fougerolles et al. and inPublished US Patent application 2012/0251618 to Schrum et al.

Additional therapeutic macromolecules useful in accordance with aspectsof this invention will be apparent to those of skill in the art, and theinvention is not limited in this respect.

In some embodiments, a component, such as a therapeutic macromolecule orimmunosuppressant, may be isolated. Isolated refers to the element beingseparated from its native environment and present in sufficientquantities to permit its identification or use. This means, for example,the element may be (i) selectively produced by expression cloning or(ii) purified as by chromatography or electrophoresis. Isolated elementsmay be, but need not be, substantially pure. Because an isolated elementmay be admixed with a pharmaceutically acceptable excipient in apharmaceutical preparation, the element may comprise only a smallpercentage by weight of the preparation. The element is nonethelessisolated in that it has been separated from the substances with which itmay be associated in living systems, i.e., isolated from other lipids orproteins. Any of the elements provided herein may be isolated andincluded in the compositions or used in the methods in isolated form.

D. Methods of Making and Using the Compositions and Related Methods

Aspects of the invention relate to determining a protocol for themethods of concomitant local administration as provided herein. Aprotocol can be determined by varying the frequency, dosage amount andother aspects of administration of the therapeutic macromolecule and thecomposition of immunosuppressant and subsequently assessing a localinflammatory response, such as Type I and Type IV hypersensitivity basedon such variation. A preferred protocol for practice of the inventionreduces or prevents local inflammation.

Synthetic nanocarriers may be prepared using a wide variety of methodsknown in the art. For example, synthetic nanocarriers can be formed bymethods such as nanoprecipitation, flow focusing using fluidic channels,spray drying, single and double emulsion solvent evaporation, solventextraction, phase separation, milling, microemulsion procedures,microfabrication, nanofabrication, sacrificial layers, simple andcomplex coacervation, and other methods well known to those of ordinaryskill in the art. Alternatively or additionally, aqueous and organicsolvent syntheses for monodisperse semiconductor, conductive, magnetic,organic, and other nanomaterials have been described (Pellegrino et al.,2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci., 30:545; andTrindade et al., 2001, Chem. Mat., 13:3843). Additional methods havebeen described in the literature (see, e.g., Doubrow, Ed.,“Microcapsules and Nanoparticles in Medicine and Pharmacy,” CRC Press,Boca Raton, 1992; Mathiowitz et al., 1987, J. Control. Release, 5:13;Mathiowitz et al., 1987, Reactive Polymers, 6:275; and Mathiowitz etal., 1988, J. Appl. Polymer Sci., 35:755; U.S. Pat. Nos. 5,578,325 and6,007,845; P. Paolicelli et al., “Surface-modified PLGA-basedNanoparticles that can Efficiently Associate and Deliver Virus-likeParticles” Nanomedicine. 5(6):843-853 (2010)).

Various materials may be encapsulated into synthetic nanocarriers asdesirable using a variety of methods including but not limited to C.Astete et al., “Synthesis and characterization of PLGA nanoparticles” J.Biomater. Sci. Polymer Edn, Vol. 17, No. 3, pp. 247-289 (2006); K.Avgoustakis “Pegylated Poly(Lactide) and Poly(Lactide-Co-Glycolide)Nanoparticles: Preparation, Properties and Possible Applications in DrugDelivery” Current Drug Delivery 1:321-333 (2004); C. Reis et al.,“Nanoencapsulation I. Methods for preparation of drug-loaded polymericnanoparticles” Nanomedicine 2:8— 21 (2006); P. Paolicelli et al.,“Surface-modified PLGA-based Nanoparticles that can EfficientlyAssociate and Deliver Virus-like Particles” Nanomedicine. 5(6):843-853(2010). Other methods suitable for encapsulating materials intosynthetic nanocarriers may be used, including without limitation methodsdisclosed in U.S. Pat. No. 6,632,671 to Unger issued Oct. 14, 2003.

In certain embodiments, synthetic nanocarriers are prepared by ananoprecipitation process or spray drying. Conditions used in preparingsynthetic nanocarriers may be altered to yield particles of a desiredsize or property (e.g., hydrophobicity, hydrophilicity, externalmorphology, “stickiness,” shape, etc.). The method of preparing thesynthetic nanocarriers and the conditions (e.g., solvent, temperature,concentration, air flow rate, etc.) used may depend on the materials tobe attached to the synthetic nanocarriers and/or the composition of thepolymer matrix.

If synthetic nanocarriers prepared by any of the above methods have asize range outside of the desired range, such synthetic nanocarriers canbe sized, for example, using a sieve.

Elements (i.e., components) of the synthetic nanocarriers may beattached to the overall synthetic nanocarrier, e.g., by one or morecovalent bonds, or may be attached by means of one or more linkers.Additional methods of functionalizing synthetic nanocarriers may beadapted from Published US Patent Application 2006/0002852 to Saltzman etal., Published US Patent Application 2009/0028910 to DeSimone et al., orPublished International Patent Application WO/2008/127532 A1 to Murthyet al.

Alternatively or additionally, synthetic nanocarriers can be attached tocomponents directly or indirectly via non-covalent interactions. Innon-covalent embodiments, the non-covalent attaching is mediated bynon-covalent interactions including but not limited to chargeinteractions, affinity interactions, metal coordination, physicaladsorption, host-guest interactions, hydrophobic interactions, TTstacking interactions, hydrogen bonding interactions, van der Waalsinteractions, magnetic interactions, electrostatic interactions,dipole-dipole interactions, and/or combinations thereof. Suchattachments may be arranged to be on an external surface or an internalsurface of a synthetic nanocarrier. In embodiments, encapsulation and/orabsorption is a form of attaching. In embodiments, the syntheticnanocarriers can be combined with a therapeutic macromolecule byadmixing in the same vehicle or delivery system.

Compositions provided herein may comprise inorganic or organic buffers(e.g., sodium or potassium salts of phosphate, carbonate, acetate, orcitrate) and pH adjustment agents (e.g., hydrochloric acid, sodium orpotassium hydroxide, salts of citrate or acetate, amino acids and theirsalts) antioxidants (e.g., ascorbic acid, alpha-tocopherol), surfactants(e.g., polysorbate 20, polysorbate 80, polyoxyethylene9-10 nonyl phenol,sodium desoxycholate), solution and/or cryo/lyo stabilizers (e.g.,sucrose, lactose, mannitol, trehalose), osmotic adjustment agents (e.g.,salts or sugars), antibacterial agents (e.g., benzoic acid, phenol,gentamicin), antifoaming agents (e.g., polydimethylsilozone),preservatives (e.g., thimerosal, 2-phenoxyethanol, EDTA), polymericstabilizers and viscosity-adjustment agents (e.g., polyvinylpyrrolidone,poloxamer 488, carboxymethylcellulose) and co-solvents (e.g., glycerol,polyethylene glycol, ethanol).

Compositions according to the invention may comprise pharmaceuticallyacceptable excipients. The compositions may be made using conventionalpharmaceutical manufacturing and compounding techniques to arrive atuseful dosage forms. Techniques suitable for use in practicing thepresent invention may be found in Handbook of Industrial Mixing: Scienceand Practice, Edited by Edward L. Paul, Victor A. Atiemo-Obeng, andSuzanne M. Kresta, 2004 John Wiley & Sons, Inc.; and Pharmaceutics: TheScience of Dosage Form Design, 2nd Ed. Edited by M. E. Auten, 2001,Churchill Livingstone. In an embodiment, compositions are in a sterilesaline solution for injection together with a preservative.

It is to be understood that the compositions of the invention can bemade in any suitable manner, and the invention is in no way limited tocompositions that can be produced using the methods described herein.Selection of an appropriate method of manufacture may require attentionto the properties of the particular moieties being associated.

In some embodiments, compositions are manufactured under sterileconditions or are terminally sterilized. This can ensure that resultingcompositions are sterile and non-infectious, thus improving safety whencompared to non-sterile compositions. This provides a valuable safetymeasure, especially when subjects receiving the compositions have immunedefects, are suffering from infection, and/or are susceptible toinfection. In some embodiments, the compositions may be lyophilized andstored in suspension or as lyophilized powder depending on theformulation strategy for extended periods without losing activity.

Administration according to the invention may be by a variety of routes,including but not limited to subcutaneous, intramuscular and intradermalroutes. The compositions referred to herein may be manufactured andprepared for administration, preferably concomitant administration,using conventional methods.

The compositions of the invention can be administered in effectiveamounts, such as the effective amounts described elsewhere herein. Dosesof dosage forms may contain varying amounts of immunosuppressants and/ortherapeutic macromolecule, according to the invention. The amount ofimmunosuppressants and/or therapeutic macromolecule present in thedosage forms can be varied according to the nature of the therapeuticmacromolecules, and/or immunosuppressants, the therapeutic benefit to beaccomplished, and other such parameters. In embodiments, dose rangingstudies can be conducted to establish optimal therapeutic amount of theimmunosuppressants and/or therapeutic macromolecules to be present indosage forms. In embodiments, the immunosuppressants and/or therapeuticmacromolecules are present in dosage forms in an amount effective togenerate a tolerogenic immune response to the therapeutic macromoleculesupon administration to a subject. In preferable embodiments, theimmunosuppressants and/or therapeutic macromolecules are present indosage forms in an amount effective to reduce Type I and Type IVhypersensitivity when concomitantly administered locally to a subject.It may be possible to determine amounts of the immunosuppressants and/ortherapeutic macromolecules effective to generate desired immuneresponses using conventional dose ranging studies and techniques insubjects. Inventive dosage forms may be administered at a variety offrequencies. In a preferred embodiment, at least one administration ofthe compositions provided herein is sufficient to generate a a desiredresponse. In more preferred embodiments, more than one administrationsis utilized to ensure a desired response.

In some embodiments, local administration of immunosuppressants, such asthose attached to synthetic nanocarriers, with a therapeuticmacromolecule is undertaken e.g., prior to subsequent further localadministration of the therapeutic macromolecule. In exemplaryembodiments, immunosuppressants, such as those attached to syntheticnanocarriers, are locally administered with concomitant, localadministration of therapeutic macromolecule prior to subsequent furtherlocal administration of the therapeutic macromolecule.

Another aspect of the disclosure relates to kits. In some embodiments,the kit comprises an immunosuppressant, in some embodiments attached tosynthetic nanocarriers, and a therapeutic dose of a therapeuticmacromolecule. The immunosuppressant and therapeutic dose of therapeuticmacromolecule can be contained within separate containers or within thesame container in the kit. In some embodiments, the container is a vialor an ampoule. In some embodiments, the therapeutic dose of therapeuticmacromolecule and/or immunosuppressant are contained within a solutionseparate from the container, such that the therapeutic dose oftherapeutic macromolecule and/or immunosuppressant may be added to thecontainer at a subsequent time. In some embodiments, the therapeuticdose of therapeutic macromolecule and/or immunosuppressant are inlyophilized form each in a separate container or in the same container,such that they may be reconstituted at a subsequent time. In someembodiments, the kit further comprises instructions for reconstitution,mixing, administration, etc. In some embodiments, the instructionsinclude a description of the methods described herein. Instructions canbe in any suitable form, e.g., as a printed insert or a label. In someembodiments, the kit further comprises one or more syringes or othermeans for locally administering the composition and therapeutic dose oftherapeutic macromolecule.

EXAMPLES Example 1: Evaluating Tolerogenic Immune Responses withSynthetic Nanocarriers Comprising Immunosuppressant In Vivo (Prophetic)Method for Synthetic Nanocarrier Containing Rapamycin

A primary water-in-oil emulsion is prepared first. W1/O1 is prepared bycombining 0.13 M hydrochloric acid solution (0.2 mL), solution 2 (0.75mL), solution 3 (0.25 mL), and solution 4 (0.2 mL) in a small pressuretube and sonicating at 50% amplitude for 40 seconds using a BransonDigital Sonifier 250. A secondary emulsion (W1/O1/W2) is then preparedby combining solution 5 (3.0 mL) with the primary W1/O1 emulsion,vortexing for 10 s, and sonicating at 30% amplitude for 60 seconds usingthe Branson Digital Sonifier 250.

The W1/O1/W2 emulsion is added to a beaker containing 70 mM pH 8phosphate buffer solution (30 mL) and stirred at room temperature for 2hours to allow the methylene chloride to evaporate and for the syntheticnanocarriers to form. A portion of the synthetic nanocarriers are washedby transferring the synthetic nanocarrier suspension to a centrifugetube and centrifuging at 21,000×g and 4° C. for one hour, removing thesupernatant, and re-suspending the pellet in phosphate buffered saline.The washing procedure is repeated, and the pellet is re-suspended inphosphate buffered saline for a final synthetic nanocarrier dispersionof about 10 mg/mL.

The amount of rapamycin in the synthetic nanocarrier is determined byHPLC analysis. The total dry-synthetic nanocarrier mass per mL ofsuspension is determined by a gravimetric method.

Method for Measuring Rapamycin Load

Approximately 3 mg of synthetic nanocarriers are collected andcentrifuged to separate supernatant from synthetic nanocarrier pellet.Acetonitrile is added to the pellet, and the sample is sonicated andcentrifuged to remove any insoluble material. The supernatant and pelletare injected on RP-HPLC and absorbance is read at 278 nm. The μg foundin the pellet are used to calculate % entrapped (load), μg insupernatant and pellet are used to calculate total μg recovered.

Measurement of IgG

The level of IgG antibodies are measured. Blocker Casein in PBS (ThermoFisher, Catalog #37528) is used as diluent. 0.05% Tween-20 in PBS isused as wash buffer, prepared by adding 10 ml of Tween-20 ((Sigma,Catalog #P9416-100 mL) to 2 liters of a 10×PBS stock (PBS: OmniPur®10×PBS Liquid Concentrate, 4L, EMD Chemicals, Catalog #6505) and 18Liters of deionized water.

Anti-TNFα at a stock concentration of 5 mg/ml is used as a coatingmaterial. A 1:1000 dilution to 5 μg/ml is used as a workingconcentration. Each well of the assay plates is coated with 100 μldiluted OVA per well, plates are sealed with sealing film (VWR catalog#60941-120), and incubated overnight at 4° C. Costar 9017 96-well Flatbottom plates are used as assay plates (Costar 9017).

Low-binding polypropylene 96-well plate or tubes are used as set-upplates, in which samples are prepared before being transferred to theassay plate. The setup plates did not contain any antigen and,therefore, serum antibodies did not bind to the plate during the setupof the samples. Setup plates are used for sample preparation to minimizebinding that might occur during preparation or pipetting of samples ifan antigen-coated plate is used to prepare the samples. Before preparingsamples in the setup plate, wells are covered with diluent to block anynon-specific binding and the plate is sealed and incubated at 4° C.overnight.

Assay plates are washed three times with wash buffer, and wash buffer iscompletely aspirated out of the wells after the last wash. Afterwashing, 300 μl diluent are added to each well of assay plate(s) toblock non-specific binding and plates are incubated at least 2 hours atroom temperature. Serum samples are prepared in the setup plate atappropriate starting dilutions. Starting dilutions are sometimes alsoprepared in 1.5 ml tubes using diluent and then transferred to theset-up plate. Appropriate starting dilutions are determined based onprevious data, where available. Where no previous data is available, thelowest starting dilution is 1:40. Once diluted, 200 μl of the startingdilution of the serum sample is transferred from the tube to theappropriate well of the setup plate.

Once all samples were prepared in the setup plate, the plate is sealedand stored at 4° C. until blocking of the assay plates is complete.Assay plates are washed three times with wash buffer, and wash buffer iscompletely aspirated after the last wash. After washing, 100 μL ofdiluent is added to wells in of the assay plates. A pipet is used totransfer samples from the setup plate to the assay plate. Samples aremixed prior to transfer by pipetting 150 μl of diluted serum up and down3 times. After mixing, 1500 of each sample is transferred from the setupplate and added to the respective assay plate.

Once the starting dilutions of each sample are transferred from thesetup plate to the assay plate, serial dilutions are pipetted on theassay plate as follows: 50 μl of each serum sample is removed using apipet and mixed with the 100 μl of diluent previously added. This stepis repeated down the entire plate. After pipetting the dilution of thefinal row, 50 μl of fluid is removed from the wells in the final row anddiscarded, resulting in a final volume of 100 μl in every well of theassay plate. Once sample dilutions are prepared in the assay plates, theplates are incubated at room temperature for at least 2 hours.

After the incubation, plates are washed three times with wash buffer.Detection antibody (Goat anti-mouse anti-IgG, HRP conjugated) is diluted1:1500 (0.33 μg/mL) in diluent and 100 μl of the diluted antibody isadded to each well. Plates are incubated for 1 hour at room temperatureand then washed three times with wash buffer, with each washing stepincluding a soak time of at least 30 seconds.

After washing, detection substrate is added to the wells. Equal parts ofsubstrate A and substrate B (BD Biosciences TMB Substrate Reagent Set,catalog #555214) are combined immediately before addition to the assayplates, and 100 μl of the mixed substrate solution are added to eachwell and incubated for 10 minutes in the dark. The reaction is stoppedby adding 50 μl of stop solution (2N H2SO4) to each well after the 10minute period. The optical density (OD) of the wells is assessedimmediately after adding the stop solution on a plate reader at 450 nmwith subtraction at 570 nm. Data analysis is performed using MolecularDevice's software SoftMax Pro v5.4. A four-parameter logistic curve-fitgraph is prepared with the dilution on the x-axis (log scale) and the ODvalue on the y-axis (linear scale), and the half maximum value (EC50)for each sample is determined. The plate template at the top of thelayout is adjusted to reflect the dilution of each sample (1 percolumn).

Example 2: Polymeric Nanocarrier Containing Polymer-Rapamycin Conjugate(Prophetic)

Preparation of PLGA-Rapamycin Conjugate:

PLGA polymer with acid end group (7525 DLG1A, acid number 0.46 mmol/g,Lakeshore Biomaterials; 5 g, 2.3 mmol, 1.0 eq) is dissolved in 30 mL ofdichloromethane (DCM). N,N-Dicyclohexylcarbodimide (1.2 eq, 2.8 mmol,0.57 g) is added followed by rapamycin (1.0 eq, 2.3 mmol, 2.1 g) and4-dimethylaminopyridine (DMAP) (2.0 eq, 4.6 mmol, 0.56 g). The mixtureis stirred at rt for 2 days. The mixture is then filtered to removeinsoluble dicyclohexylurea. The filtrate is concentrated to ca. 10 mL involume and added to 100 mL of isopropyl alcohol (IPA) to precipitate outthe PLGA-rapamycin conjugate. The IPA layer is removed and the polymeris then washed with 50 mL of IPA and 50 mL of methyl t-butyl ether(MTBE). The polymer is then dried under vacuum at 35 C for 2 days togive PLGA-rapamycin as a white solid (ca. 6.5 g).

Nanocarrier containing PLGA-rapamycin is prepared according to theprocedure described in Example 1 as follows:

Solutions for Nanocarrier Formation are Prepared as Follows:

Solution 1: PLGA-rapamycin @ 100 mg/mL in methylene chloride. Thesolution is prepared by dissolving PLGA-rapamycin in pure methylenechloride. Solution 2: PLA-PEG @ 100 mg/mL in methylene chloride. Thesolution is prepared by dissolving PLA-PEG in pure methylene chloride.Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8 phosphatebuffer.

A primary water-in-oil emulsion is prepared first. W1/O1 is prepared bycombining solution 1 (0.75 mL), and solution 2 (0.25 mL) in a smallpressure tube and sonicating at 50% amplitude for 40 seconds using aBranson Digital Sonifier 250. A secondary emulsion (W1/O1/W2) is thenprepared by combining solution 3 (3.0 mL) with the primary W1/O1emulsion, vortexing for 10 s, and sonicating at 30% amplitude for 60seconds using the Branson Digital Sonifier 250. The W1/O1/W2 emulsion isadded to a beaker containing 70 mM pH 8 phosphate buffer solution (30mL) and stirred at room temperature for 2 hours to allow the methylenechloride to evaporate and for the nanocarriers to form. A portion of thenanocarriers is washed by transferring the nanocarrier suspension to acentrifuge tube and centrifuging at 75,600×g and 4° C. for 35 min,removing the supernatant, and re-suspending the pellet in phosphatebuffered saline. The washing procedure is repeated, and the pellet isre-suspended in phosphate buffered saline for a final nanocarrierdispersion of about 10 mg/mL.

Example 3: Preparation of Gold Nanocarriers (AuNCs) Containing Rapamycin(Prophetic)

Preparation of HS-PEG-Rapamycin:

A solution of PEG acid disulfide (1.0 eq), rapamycin (2.0-2.5 eq), DCC(2.5 eq) and DMAP (3.0 eq) in dry DMF is stirred at rt overnight. Theinsoluble dicyclohexylurea is removed by filtration and the filtrate isadded to isopropyl alcohol (IPA) to precipitate out thePEG-disulfide-di-rapamycin ester and washed with IPA and dried. Thepolymer is then treated with tris(2-carboxyethyl)phosphine hydrochloridein DMF to reduce the PEG disulfide to thiol PEG rapamycin ester(HS-PEG-rapamycin). The resulting polymer is recovered by precipitationfrom IPA and dried as previously described and analyzed by H NMR andGPC.

Formation of Gold NCs (AuNCs):

An aq. solution of 500 mL of 1 mM HAuCl4 is heated to reflux for 10 minwith vigorous stirring in a 1 L round-bottom flask equipped with acondenser. A solution of 50 mL of 40 mM of trisodium citrate is thenrapidly added to the stirring solution. The resulting deep wine redsolution is kept at reflux for 25-30 min and the heat is withdrawn andthe solution is cooled to room temperature. The solution is thenfiltered through a 0.8 μm membrane filter to give the AuNCs solution.The AuNCs are characterized using visible spectroscopy and transmissionelectron microscopy. The AuNCs are ca. 20 nm diameter capped by citratewith peak absorption at 520 nm.

AuNCs Conjugate with HS-PEG-Rapamycin:

A solution of 150 μl of HS-PEG-rapamycin (10 μM in 10 mM pH 9.0carbonate buffer) is added to 1 mL of 20 nm diameter citrate-capped goldnanocarriers (1.16 nM) to produce a molar ratio of thiol to gold of2500:1. The mixture is stirred at room temperature under argon for 1hour to allow complete exchange of thiol with citrate on the goldnanocarriers. The AuNCs with PEG-rapamycin on the surface is thenpurified by centrifuge at 12,000 g for 30 minutes. The supernatant isdecanted and the pellet containing AuNC-S-PEG-rapamycin is then pelletwashed with 1×PBS buffer. The purified Gold-PEG-rapamycin nanocarriersare then resuspend in suitable buffer for further analysis andbioassays.

Example 4: Mesoporous Silica Nanoparticles with Attached Ibuprofen(Prophetic)

Mesoporous SiO2 nanoparticle cores are created through a sol-gelprocess. Hexadecyltrimethyl-ammonium bromide (CTAB) (0.5 g) is dissolvedin deionized water (500 mL), and then 2 M aqueous NaOH solution (3.5 mL)is added to the CTAB solution. The solution is stirred for 30 min, andthen Tetraethoxysilane (TEOS) (2.5 mL) is added to the solution. Theresulting gel is stirred for 3 h at a temperature of 80° C. The whiteprecipitate which forms is captured by filtration, followed by washingwith deionized water and drying at room temperature. The remainingsurfactant is then extracted from the particles by suspension in anethanolic solution of HCl overnight. The particles are washed withethanol, centrifuged, and redispersed under ultrasonication. This washprocedure is repeated two additional times.

The SiO2 nanoparticles are then functionalized with amino groups using(3-aminopropyl)-triethoxysilane (APTMS). To do this, the particles aresuspended in ethanol (30 mL), and APTMS (50 μL) is added to thesuspension. The suspension is allowed to stand at room temperature for 2h and then is boiled for 4 h, keeping the volume constant byperiodically adding ethanol. Remaining reactants are removed by fivecycles of washing by centrifugation and redispersing in pure ethanol.

In a separate reaction, 1-4 nm diameter gold seeds are created. Allwater used in this reaction is first deionized and then distilled fromglass. Water (45.5 mL) is added to a 100 mL round-bottom flask. Whilestirring, 0.2 M aqueous NaOH (1.5 mL) is added, followed by a 1% aqueoussolution of tetrakis(hydroxymethyl)phosphonium chloride (THPC) (1.0 mL).Two minutes after the addition of THPC solution, a 10 mg/mL aqueoussolution of chloroauric acid (2 mL), which has been aged at least 15min, is added. The gold seeds are purified through dialysis againstwater.

To form the core-shell nanocarriers, the amino-functionalized SiO2nanoparticles formed above are first mixed with the gold seeds for 2 hat room temperature. The gold-decorated SiO2 particles are collectedthrough centrifugation and mixed with an aqueous solution of chloroauricacid and potassium bicarbonate to form the gold shell. The particles arethen washed by centrifugation and redispersed in water. Ibuprofen isloaded by suspending the particles in a solution of sodium ibuprofen (1mg/L) for 72 h. Free ibuprofen is then washed from the particles bycentrifugation and redispersing in water.

Example 5: Liposomes Containing Cyclosporine A (Prophetic)

The liposomes are formed using thin film hydration.1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (32 mol), cholesterol(32 mol), and cyclosporin A (6.4 μmol) are dissolved in pure chloroform(3 mL). This lipid solution is added to a 50 mL round-bottom flask, andthe solvent is evaporated on a rotary evaporator at a temperature of 60°C. The flask is then flushed with nitrogen gas to remove remainingsolvent. Phosphate buffered saline (2 mL) and five glass beads are addedto the flask, and the lipid film is hydrated by shaking at 60° C. for 1h to form a suspension. The suspension is transferred to a smallpressure tube and sonicated at 60° C. for four cycles of 30s pulses witha 30 s delay between each pulse. The suspension is then left undisturbedat room temperature for 2 h to allow for complete hydration. Theliposomes are washed by centrifugation followed by resuspension in freshphosphate buffered saline.

Example 6: Synthetic Nanocarriers Containing Rapamycin Materials

Rapamycin was purchased from TSZ CHEM (185 Wilson Street, Framingham,Mass. 01702; Product Catalogue #R1017). PLGA with 76% lactide and 24%glycolide content and an inherent viscosity of 0.69 dL/g was purchasedfrom SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala.35211. Product Code 7525 DLG 7A.) PLA-PEG block co-polymer with a PEGblock of approximately 5,000 Da and PLA block of approximately 40,000 Dawas purchased from SurModics Pharmaceuticals (756 Tom Martin Drive,Birmingham, Ala. 35211; Product Code 100 DL mPEG 5000 5CE). Polyvinylalcohol (85-89% hydrolyzed) was purchased from EMD Chemicals (ProductNumber 1.41350.1001).

Method

Solutions were Prepared as Follows:

Solution 1: PLGA at 75 mg/mL and PLA-PEG at 25 mg/mL in methylenechloride. The solution was prepared by dissolving PLGA and PLA-PEG inpure methylene chloride.

Solution 2: Rapamycin at 100 mg/mL in methylene chloride. The solutionwas prepared by dissolving rapamycin in pure methylene chloride.

Solution 3: Polyvinyl alcohol at 50 mg/mL in 100 mM pH 8 phosphatebuffer.

An oil-in-water emulsion was used to prepare the nanocarriers. The O/Wemulsion was prepared by combining solution 1 (1 mL), solution 2 (0.1mL), and solution 3 (3 mL) in a small pressure tube and sonicating at30% amplitude for 60 seconds using a Branson Digital Sonifier 250. TheO/W emulsion was added to a beaker containing 70 mM pH 8 phosphatebuffer solution (30 mL) and stirred at room temperature for 2 hours toallow the methylene chloride to evaporate and for the nanocarriers toform. A portion of the nanocarriers was washed by transferring thenanocarrier suspension to a centrifuge tube and centrifuging at 75,000×gand 4° C. for 35 min, removing the supernatant, and re-suspending thepellet in phosphate buffered saline. The washing procedure was repeated,and the pellet was re-suspended in phosphate buffered saline for a finalnanocarrier dispersion of about 10 mg/mL.

Nanocarrier size was determined by dynamic light scattering. The amountrapamycin in the nanocarrier was determined by HPLC analysis. The totaldry-nanocarrier mass per mL of suspension was determined by agravimetric method.

Effective Diameter Rapamycin Content (nm) (% w/w) 227 6.4

Example 7: Evaluating Tolerogenic Immune Responses FollowingConcomittant Administration of Synthetic Nanocarriers ComprisingImmunosuppressant and Therapeutic Proteins

Age-matched (5 weeks) C57BL/6 female mice were injected with 60 μg, 20μg, 6 μg or 0.2 μg (the doses of 20 μg, 6 μg and 0.2 μg aresubtherapeutic doses, while 60 μg is a therapeutic dose) of theanti-TNFα antibody HUMIRA s.c. in the hind limbs. Control groups wereleft untreated (No Treatment) whereas three other groups where treatedwith synthetic nanocarriers comprising Rapamycin (containing 100 μg ofRapamycin). In one group the synthetic nanocarriers were admixed withthe same injection (d0), another group received the syntheticnanocarriers in the same site one day prior to HUMIRA injection (d−1)while the last group received an injection of synthetic nanocarriersadmixed with 15 ng of HUMIRA 7 days prior to challenge (d−7) with the 60μg dose, 20 μg, or 6 μg dose. After this one-time treatment, all animalsreceived another injection on day 7, 14, 22 and 29. The antibody titerswere assessed in the blood from these animals collected at day 21. Onthe last challenge the local inflammation caused by the hypersensitivityto HUMIRA was monitored by measuring the ventral-dorsal hind limbthickness 40 minutes after the injection with a caliper. One limb wasinjected with HUMIRA while the other limb was injected with saline. Theresults are expressed as the difference in thickness between the twohind limbs. Interestingly, the largest reductions in antibody titerswere seen with doses that were at least equal to the therapeutic dosewith less of a reduction seen in subtherapeutic doses (FIGS. 1 and 2 ).

These results show that compositions provided herein when administeredconcomitantly with a therapeutic macromolecule can reduce formation ofType I hypersensitivity or Type 1V hypersensitivity in a subject.

Example 8: Reduction of KLH Hypersensitivity with Tolerogenic SyntheticNanocarriers Materials

Rapamycin was purchased from TSZ CHEM (185 Wilson Street, Framingham,Mass. 01702; Product Code R1017). PLGA with a lactide:glycolide ratio of3:1 and an inherent viscosity of 0.75 dL/g was purchased from SurModicsPharmaceuticals (756 Tom Martin Drive, Birmingham, Ala. 35211; ProductCode 7525 DLG 7A). PLA-PEG-OMe block co-polymer with a methyl etherterminated PEG block of approximately 5,000 Da and an overall inherentviscosity of 0.5 DL/g was purchased from Lakeshore Biochemicals (756 TomMartin Drive, Birmingham, Ala. 35211; Product Code 100 DL mPEG 50005CE). EMPROVE® Polyvinyl Alcohol 4-88, USP (85-89% hydrolyzed, viscosityof 3.4-4.6 mPa s) was purchased from EMD Chemicals Inc. (480 SouthDemocrat Road Gibbstown, N.J. 08027. Product Code 1.41350).

Method

Solutions were Prepared as Follows:

Solution 1: PLGA at 75 mg/mL, PLA-PEG-OMe at 25 mg/mL, and rapamycin at12.5 mg/mL in methylene chloride. The solution was prepared bydissolving PLGA, PLA-PEG-OMe, and rapamycin in pure methylene chloride.Solution 2: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8 phosphatebuffer.

An oil-in-water emulsion was used to prepare the nanocarriers. The 01Wemulsion was prepared by combining Solution 1 (1.0 mL) and Solution 2(3.0 mL) in a small pressure tube and sonicating at 30% amplitude for 60seconds using a Branson Digital Sonifier 250. The 01W emulsion was addedto a beaker containing 70 mM pH 8 phosphate buffer solution and stirredat room temperature for 2 hours to allow the methylene chloride toevaporate and for the nanocarriers to form. A portion of thenanocarriers was washed by transferring the nanocarrier suspension to acentrifuge tube and centrifuging at 75,600×g and 4° C. for 50 min,removing the supernatant, and re-suspending the pellet in phosphatebuffered saline. The washing procedure was repeated, and the pellet wasre-suspended in phosphate buffered saline for a final nanocarrierdispersion of about 10 mg/mL.

Nanocarrier size was determined by dynamic light scattering. The amountof rapamycin in the nanocarrier was determined by HPLC analysis. Thetotal dry-nanocarrier mass per mL of suspension was determined by agravimetric method.

Effective Diameter Rapamycin Content (nm) (% w/w) 241 11.5

Control C57BL/6 age-matched (5-6 weeks) females were injected s.c. inthe hind limbs with 5 μg of KLH and 20 μg of CpG ODN. Another groupreceived the same mixture but 0.43 mg of tolerogenic syntheticnanocarriers containing rapamycin (tSIP, NP[Rapa]) were admixed. After 5days all animals received a challenge with KLH (50 μg) in one hind limbwhile the other received saline to test the local T cell-mediated TypeIV hypersensitivity responses.

For this, the thickness of the hind limbs were measured with the help ofa caliper 3, 24, 48 and 72 hours after the injection. The difference inthickness between the two limbs was plotted in FIG. 3 (left panel).Untreated control animals experience a mounting inflammatory responsestarting 3 hours after administration of KLH and peaking at 48 hours.The treatment with tolerogenic synthetic nanocarriers led to a higherbut transient inflammatory response 3 hours after the challenge but itdissipated quickly the next day. These results correlated with the levelof the anti-KLH antibody response found in the blood of these animals onday 11 (FIG. 3 right panel.

These results show that compositions provided herein when administeredconcomitantly with a therapeutic macromolecule can reduce formation ofimmune responses that can give rise to adverse site reactions, such asType IV hypersensitivity or Type I hypersensitivity reactions.

Example 9: Antigen-Specific Tolerogenic Responses to Chicken Ovalbuminwith Encapsulated Rapamycin NP[Rapa] Materials and Methods Materials

Rapamycin was purchased from TSZ CHEM (185 Wilson Street, Framingham,Mass. 01702), product code R1017. PLGA with a lactide:glycolide ratio of1:1 and an inherent viscosity of 0.24 dL/g was purchased from LakeshoreBiomaterials (756 Tom Martin Drive, Birmingham, Ala. 35211), productcode 5050 DLG 2.5A. PLA-PEG-OMe block co-polymer with a methyl etherterminated PEG block of approximately 5,000 Da and an overall inherentviscosity of 0.50 DL/g was purchased from Lakeshore Biomaterials (756Tom Martin Drive, Birmingham, Ala. 35211), product code 100 DL mPEG 50005CE. EMPROVE® Polyvinyl Alcohol 4-88, USP (85-89% hydrolyzed, viscosityof 3.4-4.6 mPa s) was purchased from EMD Chemicals Inc. (480 SouthDemocrat Road Gibbstown, N.J. 08027), product code 1.41350. Cellgrophosphate buffered saline 1×(PBS 1×) was purchased from Corning (9345Discovery Blvd. Manassas, Va. 20109), product code 21-040-CV.

Method

Solutions were Prepared as Follows:

Solution 1: A polymer and rapamycin mixture was prepared by dissolvingPLGA at 75 mg per 1 mL, PLA-PEG-Ome at 25 mg per 1 mL, and rapamycin as12.5 mg per 1 mL in dichloromethane. Solution 2: Polyvinyl alcohol wasprepared at 50 mg/mL in 100 mM pH 8 phosphate buffer.

An O/W emulsions was prepared by combining Solution 1 (1.0 mL) andSolution 2 (3.0 mL) in a small glass pressure tube and sonicating at 30%amplitude for 60 seconds using a Branson Digital Sonifier 250. The O/Wemulsion was added to an open beaker containing 70 mM pH 8 phosphatebuffer solution (60 mL). Three additional, identical O/W emulsions wereprepared and added to the same beaker as the first. These were thenstirred at room temperature for 2 hours to allow the dichloromethane toevaporate and for the nanocarriers to form. A portion of thenanocarriers was washed by transferring the nanocarrier suspension tocentrifuge tubes and centrifuging at 75,600×g and 4° C. for 35 minutes,removing the supernatant, and re-suspending the pellet in PBS 1×. Thewash procedure was repeated and then the pellet was re-suspended in PBS1× to achieve a nanocarrier suspension having a nominal concentration of10 mg/mL on a polymer basis. An identical formulation was prepared asabove in a separate beaker, and combined with the first after the washstep. The mixed nanocarrier solution was then filtered using 1.2 μm PESmembrane syringe filters from Pall part number 4656, and stored at −20°C.

Nanocarrier size was determined by dynamic light scattering. The amountof rapamycin in the nanocarrier was determined by HPLC analysis. Thetotal dry-nanocarrier mass per mL of suspension was determined by agravimetric method.

Effective Diameter Rapamycin Content (nm) (% w/w) 220 11.85

NP[OVA] Materials and Methods Materials

Ovalbumin protein, was purchased from Worthington BiochemicalCorporation (730 Vassar Avenue, Lakewood, N.J. 08701), product codeLS003054). PLGA with 54% lactide and 46% glycolide content and aninherent viscosity of 0.24 dL/g was purchased from LakeshoreBiomaterials (756 Tom Martin Drive, Birmingham, Ala. 35211), productcode 5050 DLG 2.5A). PLA-PEG block co-polymer with a methyl etherterminated PEG block of approximately 5,000 Da and Mw of 28,000 Da,inherent viscosity of 0.38 dL/g was purchased from LakeshoreBiomaterials (756 Tom Martin Drive, Birmingham, Ala. 35211), productcode 100 DL mPEG 5000 4CE. EMPROVE® Polyvinyl Alcohol 4-88, USP, 85-89%hydrolyzed, viscosity of 3.4-4.6 mPa·s, was purchased from EMD ChemicalsInc. (480 South Democrat Road Gibbstown, N.J. 08027), product code1.41350.1001. Cellgro Phosphate-buffered saline 1× (PBS 1×) waspurchased from Corning (9345 Discovery Blvd. Manassas, Va. 20109),product code 21-040-CV.

Method

Solutions were Prepared as Follows:

Solution 1: Ovalbumin protein @ 50 mg/mL was prepared in 10 mM phosphatebuffer pH 8 with 10% by weight sucrose. Solution 2: PLGA was prepared bydissolving PLGA at 100 mg per 1 mL of dichloromethane in the chemicalfume hood. Solution 3: PLA-PEG-OMe was prepared by dissolvingPLA-PEG-OMe at 100 mg per 1 mL of dichloromethane in the chemical fumehood. Solution 4: Polyvinyl alcohol @ 65 mg/mL in 100 mM phosphatebuffer, pH 8.

A primary (W1/O) emulsion was first created by mixing Solutions 1through 3. Solution 1 (0.2 mL), Solution 2 (0.75 mL), and Solution 3(0.25 mL) were combined in a small glass pressure tube which waspre-chilled >4 minutes in an ice water bath, and sonicated at 50%amplitude for 40 seconds over an ice bath using a Branson DigitalSonifier 250. A secondary (W1/O/W2) emulsion was then formed by addingSolution 4 (3 mL) to the primary emulsion, vortex mixing to create amilky dispersion, and then sonicating at 30% amplitude for 60 secondsover an ice bath using the Branson Digital Sonifier 250. The secondaryemulsion was added to an open 50 mL beaker containing PBS 1× (30 mL). Asecond identical double emulsion formulation was prepared as describedabove, and added to the same 50 mL beaker as the first. The twopreparations were stirred at room temperature for 2 hours to allow thedichloromethane to evaporate and the nanocarriers to form in suspension.A portion of the suspended nanocarriers was washed by transferring thenanocarrier suspension to a centrifuge tube, spinning at 75,600 rcf for50 minutes, removing the supernatant, and re-suspending the pellet inPBS 1×. This washing procedure was repeated and then the pellet wasre-suspended in PBS 1× to achieve a nanocarrier suspension having anominal concentration of 10 mg/mL on a polymer basis. The suspension wasstored frozen at −20C until use.

Effective Diameter Ovalbumin Content (nm) (% w/w) 164 5.81

NP[GSK1059615] Materials and Methods Materials

GSK1059615 was purchased from MedChem Express (11 Deer Park Drive, Suite102D Monmouth Junction, N.J. 08852), product code HY-12036. PLGA with alactide:glycolide ratio of 1:1 and an inherent viscosity of 0.24 dL/gwas purchased from Lakeshore Biomaterials (756 Tom Martin Drive,Birmingham, Ala. 35211), product code 5050 DLG 2.5A. PLA-PEG-OMe blockco-polymer with a methyl ether terminated PEG block of approximately5,000 Da and an overall inherent viscosity of 0.26 DL/g was purchasedfrom Lakeshore Biomaterials (756 Tom Martin Drive, Birmingham, Ala.35211; Product Code 100 DL mPEG 5000 5K-E). Cellgro phosphate bufferedsaline 1× pH 7.4 (PBS 1×) was purchased from Corning (9345 DiscoveryBlvd. Manassas, Va. 20109), product code 21-040-CV.

Method

Solutions were Prepared as Follows:

Solution 1: PLGA (125 mg), and PLA-PEG-OMe (125 mg), were dissolved in10 mL of acetone. Solution 2: GSK1059615 was prepared at 10 mg in 1 mLof N-methyl-2-pyrrolidinone (NMP).

Nanocarriers were prepared by combining Solution 1 (4 mL) and Solution 2(0.25 mL) in a small glass pressure tube and adding the mixture dropwise to a 250 mL round bottom flask containing 20 mL of ultra-pure waterunder stirring. The flask was mounted onto a rotary evaporation device,and the acetone was removed under reduced pressure. A portion of thenanocarriers was washed by transferring the nanocarrier suspension tocentrifuge tubes and centrifuging at 75,600 rcf and 4° C. for 50minutes, removing the supernatant, and re-suspending the pellet in PBS1×. The washing procedure was repeated, and the pellet was re-suspendedin PBS 1× to achieve a nanocarrier suspension having a nominalconcentration of 10 mg/mL on a polymer basis. The washed nanocarriersolution was then filtered using 1.2 μm PES membrane syringe filtersfrom Pall, part number 4656. An identical nanocarrier solution wasprepared as above, and pooled with the first after the filtration step.The homogenous suspension was stored frozen at −20° C.

Nanocarrier size was determined by dynamic light scattering. The amountof GSK1059615 in the nanocarrier was determined by UV absorption at 351nm. The total dry-nanocarrier mass per mL of suspension was determinedby a gravimetric method.

Effective Diameter GSK1059615 Content (nm) (% w/w) 143 1.02

C57BL/6 age-matched (5-6 weeks) female mice were injected i.v. in thetail vein on days −21 and −14 with saline (No Treatment), 1.1 mg ofwhole Ovalbumin-loaded nanocarriers (NP[OVA]) combined to either 1.2 mgof rapamycin-containing nanocarriers (NP[Rapa]) or 8 mg ofGSK1059615-loaded nanocarriers (NP[GSK1059615]).

At day 0 all animals were injected s.c. in the hind limbs with 25 μg ofparticulate OVA (pOVA) admixed to 2 μg of CpG followed by injections ofjust 25 μg pOVA on days 7 and 14. Antibody titers were measured on day21. In absence of any treatment, the animals developed a robust immuneresponse against OVA that can be measured by the anti-OVA IgG antibodytiters. The antibody titers at day 21 shown in FIG. 4 demonstrate that 2doses of synthetic tolerogenic nanocarriers administered concomitantlywith encapsulated OVA in the same solution (NP[OVA]+NP[Rapa] orNP[GSK1059615]) were effective in reducing antibody formation to OVAeven after 1 injection of OVA+CpG and 2 injections of OVA alone. Theseresults show that encapsulated immunosuppressants (such as rapamycin andGSK1059615]) when concomitantly delivered with a protein can preventantibody formation to that protein for multiple challenges and periodsof time.

Example 10: Local Administration of Therapeutic Doses of HUMIRA(Prophetic)

Three thousand two hundred human subjects suffering from rheumatoidarthritis are recruited for a series of clinical trials. In a pilot doseranging trial, 1200 subjects are divided into four arms (placebo and 3different doses of synthetic nanocarriers, prepared according to Example5). Each subject in each of the four arms receives two rounds of HUMIRA40 mg s.c. concomitantly with either s.c. placebo or syntheticnanocarrier. The synthetic nanocarrier dose that most reduces the meanlevel of anti-HUMIRA antibodies in an arm is declared to be theimmunosuppressant dose.

In another pilot trial, the recruited human subjects are divided into 4Test Arms of 500 subjects each. Placebo, HUMIRA, and the syntheticnanocarriers are administered concomitantly (except for Test Arm 1)according to the following table, with the synthetic nanocarriers beingadministered at the immunosuppressant dose.

Test Arm Number HUMIRA dose NC Admin (sc) 1 40 mg sc − 2 40 mg sc + 3 50mg sc + 4 30 mg sc + 5 Placebo Placebo

Local inflammation at the injection site is noted and scored using anapplicable rating scale. The mean local inflammation score is noted foreach arm. In an application of the information established during thepilot trials, one or more of the therapeutic doses of HUMIRA areadministered concomitantly with the immunosuppressant dose containingthe synthetic nanocarriers to subjects diagnosed with rheumatoidarthritis and at risk of suffering local inflammation from thetherapeutic doses of HUMIRA.

In a further embodiment, a protocol using the information establishedduring the pilot trials is prepared to guide concomitant dosing ofHUMIRA and the synthetic nanocarriers to human subjects diagnosed withrheumatoid arthritis and at risk of suffering local inflammation fromthe therapeutic doses of HUMIRA. This protocol is then used to guideconcomitant administration of therapeutic doses of HUMIRA, and thesynthetic nanocarriers, to human subjects.

Example 11: Local Administration of Therapeutic Doses of HUMIRA(Prophetic)

Three thousand two hundred human subjects suffering from rheumatoidarthritis are recruited for a series of clinical trials. In a pilot doseranging trial, 1200 subjects are divided into four arms (placebo and 3different doses of the synthetic nanocarriers of NP[GSK1059615] ofExample 9. Each subject in each of the four arms receives two rounds ofHUMIRA 40 mg s.c. concomitantly with either s.c. placebo or syntheticnanocarrier. The synthetic nanocarrier dose that most reduces the meanlevel of anti-HUMIRA antibodies in an arm is declared to be theimmunosuppressant dose.

In another pilot trial, the recruited human subjects are divided into 4Test Arms of 500 subjects each. Placebo, HUMIRA, and the syntheticnanocarriers are administered concomitantly (except for Test Arm 1)according to the following table, with the synthetic nanocarriers beingadministered at the immunosuppressant dose.

Test Arm Number HUMIRA dose NC Admin (sc) 1 40 mg sc − 2 40 mg sc + 3 50mg sc + 4 30 mg sc + 5 Placebo Placebo

Local inflammation at the injection site is noted and scored using anapplicable rating scale. The mean local inflammation score is noted foreach arm. In an application of the information established during thepilot trials, one or more of the therapeutic doses of HUMIRA areadministered concomitantly with the immunosuppressant dose containingthe synthetic nanocarriers to subjects diagnosed with rheumatoidarthritis and at risk of suffering local inflammation from thetherapeutic doses of HUMIRA.

In a further embodiment, a protocol using the information establishedduring the pilot trials is prepared to guide concomitant dosing ofHUMIRA and the synthetic nanocarriers to human subjects diagnosed withrheumatoid arthritis and at risk of suffering local inflammation fromthe therapeutic doses of HUMIRA. This protocol is then used to guideconcomitant administration of therapeutic doses of HUMIRA, and thesynthetic nanocarriers, to human subjects.

Example 12: Local Administration of Therapeutic Doses of TherapeuticMacromolecule (Prophetic)

Three thousand two hundred human subjects suffering fromchemotherapy-related anemia are recruited for a series of clinicaltrials. In a pilot dose ranging trial, modified mRNR encodingerythropoietin is prepared according to US Patent application2013/0115272 to de Fougerolles et al. (“mmRNA”). Twelve hundred subjectsare divided into four arms (placebo and 3 different doses of thesynthetic nanocarriers of Example 6). Each subject in each of the fourarms receives a therapeutic dose of mmRNA concomitantly with eitherplacebo or synthetic nanocarrier. The synthetic nanocarrier dose thatmost reduces the mean level of anti-mmRNA antibodies in an arm isdeclared to be the immunosuppressant dose.

In another pilot trial, the recruited human subjects are divided into 4Test Arms of 500 subjects each. Placebo, mmRNA, and the syntheticnanocarriers are administered concomitantly (except for Test Arm 1)according to the following table, with the synthetic nanocarriers beingadministered at the immunosuppressant dose.

Test Arm Number mmRNA dose NC Admin (sc) 1 Therapeutic dose sc − 2Therapeutic dose sc + 3 1.5x Therap. dose sc + 4 0.5x Therap. dose sc +5 Placebo Placebo

Local inflammation at the injection site is noted and scored using anapplicable rating scale. The mean local inflammation score is noted foreach arm. In an application of the information established during thepilot trials, one or more of the therapeutic doses of mmRNA areadministered concomitantly with the immunosuppressant dose containingthe synthetic nanocarriers to subjects diagnosed with rheumatoidarthritis and at risk of suffering local inflammation from thetherapeutic doses of mmRNA.

In a further embodiment, a protocol using the information establishedduring the pilot trials is prepared to guide concomitant dosing of mmRNAand the synthetic nanocarriers to human subjects diagnosed withrheumatoid arthritis and at risk of suffering local inflammation fromthe therapeutic doses of mmRNA. This protocol is then used to guideconcomitant administration of therapeutic doses of mmRNA, and thesynthetic nanocarriers, to human subjects.

Example 13: Antigen-Specific Tolerogenic Responses to Chicken Ovalbuminwith Nanocrytalline Rapamycin (Prophetic)

C57BL/6 age-matched (5-6 weeks) female mice are injected i.v. in thetail vein on days −21 and −14 with saline (No Treatment) or 1.1 mg ofwhole Ovalbumin and 1.2 mg of nanocrystalline rapamycin. At day 0 allanimals are injected s.c. in the hind limbs with 25 μg of particulateOVA (pOVA) admixed to 2 μg of CpG followed by injections of just 25 μgpOVA on days 7 and 14. Antibody titers are measured on day 21. Inabsence of any treatment, the animals develop a robust immune responseagainst OVA that can be measured by the anti-OVA IgG antibody titers.

A reduction in an undesired immune response in the animals that receivedOVA in combination with nanocrystalline rapamycin indicates that thenanocrystal-form of the immunosuppressant when concomitantly deliveredwith a protein can prevent an undesired immune response to that protein.

1. A method comprising: providing a therapeutic dose of a therapeuticmacromolecule, wherein the therapeutic macromolecule is not attached tosynthetic nanocarriers; providing a composition comprising syntheticnanocarriers that are attached to immunosuppressants; and administeringthe composition and the therapeutic dose of the therapeuticmacromolecule to a subject concomitantly, wherein the concomitantadministration of the composition and the therapeutic dose of thetherapeutic macromolecule reduces both Type 1 hypersensitivity and TypeIV hypersensitivity in the subject.
 2. The method of claim 1, whereinthe subject is a naïve subject.
 3. The method of claim 1, wherein thecomposition and the therapeutic dose of the therapeutic macromoleculeare administered to the same location.
 4. The method of any claim 1,wherein the composition and the therapeutic dose of the therapeuticmacromolecule are administered to different locations.
 5. The method ofclaim 1, wherein the concomitant administration is according to aprotocol that has been demonstrated to result in a reduction of bothType 1 hypersensitivity and Type IV hypersensitivity with thecomposition and therapeutic dose of the therapeutic macromolecule, ascompared to administration of the therapeutic dose of the therapeuticmacromolecule in the absence of concomitant administration of thecomposition.
 6. The method of claim 5, wherein the method furthercomprises determining the protocol.
 7. The method of claim 1, whereinthe method further comprises assessing an inflammatory response in thesubject prior to and/or after the administration.
 8. The method of claim7, wherein the method further comprises assessing Type 1hypersensitivity and Type IV hypersensitivity in the subject prior toand/or after the administration.
 9. (canceled)
 10. The method of claim1, wherein the method further comprises recording a reduction orprevention of an inflammatory response.
 11. The method of claim 10,wherein the method further comprises recording a reduction in both Type1 hypersensitivity and Type IV hypersensitivity.
 12. The method of claim1, wherein the immunosuppressant comprises a statin, an mTOR inhibitor,a TGF-β signaling agent, a corticosteroid, an inhibitor of mitochondrialfunction, a P38 inhibitor, an NF-κB inhibitor, an adenosine receptoragonist, a prostaglandin E2 agonist, a phosphodiesterase 4 inhibitor, anHDAC inhibitor or a proteasome inhibitor. 13-15. (canceled)
 16. Themethod of claim 1, wherein the therapeutic macromolecule comprises a/aninfusible or injectable therapeutic protein, enzyme, enzyme cofactor,hormone, blood or blood coagulation factor, cytokine, interferon, growthfactor, monoclonal antibody, polyclonal antibody, or protein associatedwith Pompe's disease. 17-22. (canceled)
 23. The method of claim 1,wherein a load of immunosuppressant attached to the syntheticnanocarriers, on average across the synthetic nanocarriers, is between0.1% and 50%.
 24. (canceled)
 25. The method of claim 1, wherein thesynthetic nanocarriers comprise lipid nanoparticles, polymericnanoparticles, metallic nanoparticles, surfactant-based emulsions,dendrimers, buckyballs, nanowires, virus-like particles or peptide orprotein particles. 26-29. (canceled)
 30. The method of claim 25, whereinthe synthetic nanocarriers comprise polymeric nanoparticles.
 31. Themethod of claim 30, wherein the polymeric nanoparticles comprise polymerthat is a non-methoxy-terminated, pluronic polymer.
 32. The method ofclaim 30, wherein the polymeric nanoparticles comprise a polyester,polyester attached to a polyether, polyamino acid, polycarbonate,polyacetal, polyketal, polysaccharide, polyethyloxazoline orpolyethyleneimine.
 33. The method of claim 32, wherein the polyestercomprises a poly(lactic acid), poly(glycolic acid),poly(lactic-co-glycolic acid) or polycaprolactone. 34-35. (canceled) 36.The method of claim 1, wherein the mean of a particle size distributionobtained using dynamic light scattering of the synthetic nanocarriers isa diameter greater than 100 nm. 37-40. (canceled)
 41. The method ofclaim 1, wherein an aspect ratio of the synthetic nanocarriers isgreater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7 or 1:10.